Antimicrobial peptides with alpha-core helices

ABSTRACT

The present disclosure describes the identification of a consensus formula representing α-helical antimicrobial peptides (AHAPs) from broad classes of higher eukaryotes. Further provided are microbicidal peptides, compositions, methods, and uses, and computer systems and methods for identifying consensus formulae and for searching microbicidal peptides. In some embodiments, the peptide or fusion peptide includes one or more non-natural amino acid residues. Also provided is a composition comprising the α-helical antimicrobial peptide or the fusion peptide, and a pharmaceutically acceptable carrier. Also provided is a method of treating an infection in a patient in need thereof, comprising administering to the patient an effective amount of a composition comprising an α-helical antimicrobial peptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/505,013, filed May 11, 2017, the content of which is incorporated by reference in its entirety into the present disclosure.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 9, 2018, is named 254302WO_ST25.txt and is 2,033,153 bytes in size.

BACKGROUND

Antimicrobial host defense peptides (AHDPs) are an evolutionarily ancient arm of host defense that first arose in prokaryotes as a means to neutralize microbial competitors. Subsequently, similar peptides evolved in all classes of eukaryotes where they continue to act as a first line of defense against microbial invaders. AHDPs from nearly all organisms are typically small in size, cationic and amphipathic, properties that are thought to be essential for their microbicidal activities. Cationicity is thought to confer selectivity towards microorganisms, given their relatively electronegative surface charge and membrane potential. Amphipathicity has been shown to be an important feature by which AHDPs can successfully associate with and permeabilize target microbial membranes.

Given that eukaryotic AHDPs act on rapidly-evolving bacterial targets, they must necessarily react in kind to retain their potency. This host-microbe arms race has led to positive selective pressure allowing for a very high degree of mutational tolerance within AHAPs, which have been shown to be some of the most rapidly evolving sequences studied to date. When compounded over an evolutionary time scale, this process has generated an exceptionally diverse repertoire of eukaryotic sequences and structures capable of exerting microbicidal effects.

The inherent diversity in eukaryotic AHDPs has made the identification of common microbicidal motifs and SARs elusive. While a number of research groups have utilized computational and/or QSAR methods in an attempt to characterize such motifs, they have largely been focused on identifying improved drug candidates. As a result, while these investigations have identified numerous optimized or improved peptide-based therapeutics, the unifying physicochemical and three-dimensional features that confer microbicidal activity to native peptides have yet to be fully defined.

SUMMARY

The present disclosure describes the identification of a consensus formula representing α-helical antimicrobial peptides (AHAPs) from broad classes of higher eukaryotes. When this formula is applied as a component of a logical search method against proteomic databases, it consistently retrieves a majority of the known AHAP families. Furthermore, this consensus formula helped identify a number of putative novel microbicidal peptides, as well as covert antimicrobial activities within proteins for which no such activity has yet been assigned. In accordance with one embodiment of the present disclosure, therefore, provided are microbicidal peptides, compositions, methods, and uses, and computer systems and methods for identifying consensus formulae and for searching microbicidal peptides.

In one embodiment, the present disclosure provides a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14, and an amino acid derived from anyone of SEQ ID NO: 1-14 with one amino acid substitution, wherein the peptide is not longer than 100 amino acid residues in length.

In some embodiments, the peptide has antimicrobial activity. In some embodiments, the peptide comprises the amino acid sequence of anyone of SEQ ID NO: 1-14. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 13 or 14.

In some embodiments, the peptide is not longer than 75 amino acid residues in length. In some embodiments, the peptide is not longer than 60 amino acid residues in length.

Also provided, in some embodiments, is a peptide comprising an amino acid sequence of SEQ ID NO:19-6860 or an amino acid derived from a sequence of SEQ ID NO:19-6860 with one amino acid substitution, wherein the peptide is not longer than 100 amino acid residues in length.

Also provided, in some embodiments, is a fusion peptide comprising a first fragment selected from the sequences of SEQ ID NO:19-6860 or an amino acid derived from a sequence of SEQ ID NO:19-6860 with one amino acid substitution, and a second fragment having antimicrobial activity, wherein the fusion peptide is not longer than 100 amino acid residues in length. In some embodiments, the second fragment comprises a gamma-core motif comprising two anti-parallel β-sheets interposed by a short turn region with a GXC or CXG sequence pattern integrated into one of the β-sheets. In some embodiments, the gamma-core motif comprises CPTAQLIATLKNGRKICLDLQ (SEQ ID NO: 15) or a first amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15.

In some embodiments, the peptide or fusion peptide includes one or more non-natural amino acid residues.

Also provided, in one embodiment, is a composition comprising the peptide or the fusion peptide and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an antimicrobial agent. In some embodiments, the antimicrobial agent is selected from the group consisting of imipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycin and daptomycin.

Also provided, is one embodiment, is a method of treating an infection in a patient in need thereof, comprising administering to the patient an effective amount of the composition. In some embodiments, the infection is caused by a Gram-negative bacterium, a Gram-positive bacterium or a fungus.

Computer-implemented methods are also provided. In one embodiment, a method of identifying a peptide having antimicrobial activity is provided, comprising: identifying a consensus formula from aligned amino acid sequences known to have an antimicrobial activity; tuning the consensus formula with a test search against a plurality of proteins with known antimicrobial activity; and searching in a protein database, with one or more processors, for amino acid fragments matching the consensus formula, wherein the search takes as input one or more criteria selected from the group consisting of location of the fragment in a protein, size of the protein, organism of the protein, and signal peptide of the protein.

In some embodiments, the tuning comprising shortening the length of the consensus formula or changing substation options at one or more amino acid residues.

In another embodiment, a computer-implemented method of identifying an α-helical antimicrobial peptide is provided, comprising: searching in a protein database, with one or more processors, for amino acid fragments matching a consensus formula: X-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-X-[KRHEDNQSTAG]-[VILMCFWYAG], wherein X denotes any amino acid residue; filtering the searched fragments based on presence of a signal peptide in the respective protein; and evaluating the searched fragments for one or more criteria selected from the group consisting of: hydrophobic moment; mean hydrophobicity; net charge; frequencies or ratio of K and R; and isoelectric point.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain features of various embodiments of the present technology are set forth with particularity in the appended claims. A better understanding of the features and advantages of the technology will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the technology are utilized, and the accompanying drawings of which:

FIGS. 1A and 1B show graphic representations of α-core sequence formula. 1A. Helical wheel depiction of the 18 residue α-core sequence formula. 1B. Schematic representing linear formula. Hydrophobic residues (F, W, Y, V, I, L, M, C, A, G) are in various shades of green, with greater hydrophobicity indicated by increasingly darker hues. Hydrophilic residues (K, R, H, E, D, N, Q, S, T, A, G) are represented by: blue—cationic; red—anionic; orange—uncharged polar. Alanine (light green) and glycine (yellow) are included with both hydrophilic and hydrophobic groups. H—hydrophobic; P—polar.

FIG. 2 shows the alignment of prototypic AHAPs with α-core sequence formula. 18 initiation points for the scanning iterative α-core formula are shown, representing the process by which the ProSite pattern search tool was utilized to query the SwissProt database. Coloration is as per FIG. 1A-1B.

FIG. 3 illustrates the iterative optimization of the α-core formula. Iterative refinement of the α-core formula was carried out to assess the requirement for glycine and/or alanine as a component of either the polar (hydrophilic) or non-polar (hydrophobic) residue set. Percentage of returned sequences from a control AHAP dataset of more than 400 peptides are shown.

FIG. 4 illustrates a process of identifying new AHAP sequences.

FIG. 5 shows the positional and spatial amphipathic residue frequency by class. Percentages of individual residues on either the polar or non-polar peptide face of study peptides are represented as various color blocks. Residues above the x-axis are found on the polar face of retrieved peptides and residues below the axis are found on the non-polar face. Panel A. Arthropods. Panel B. Amphibians. Panel C. Higher Vertebrates.

FIG. 6 shows the comparison of N_(K)/N_(K)+N_(K) ratio and hydrophobicity in AHAP and toxin helices. Percentage of lysine (N_(K)) relative to arginine (N_(R)) expressed as (N_(K)/N_(K)+N_(K)) versus hydrophobicity (H) in study AHAPs and toxins. Preference of lysine as compared to arginine is reflected in an increased value of H for peptides capable of generating NGC in membranes as predicted by the saddle-splay rule.

FIG. 7 shows the mapping of net charge vs. hydrophobic moment in study peptides. Values for net charge (Q) versus hydrophobic moment (μH) are shown for the retrieved peptide dataset. All retrieved sequences are shown in gray. Peptide groups selected for further characterization are shown in color. For comparison, prototypic AHAPs are shown in pink.

FIG. 8 shows helical wheel depiction of study test peptides. Hydrophobic moment (μH) and vector angle direction are indicated. Coloration: cationic full charge (KR)—blue, partial charge (H)—light blue; anionic—red; polar—yellow; tiny—gray; polar (NQ)—pink, (TS)—purple.

FIG. 9 shows the antimicrobial activity of study test peptides. Microbicidal activity of study test peptides versus a panel of prototypic gram-positive (S. aureus), gram-negative (S. typhimurium, P. aeruginosa, A. baumannii) and fungal (C. albicans) pathogens at two pH's representing native physiologic (pH 7.5) or phagolysosomal (pH 5.5) environments.

FIG. 10 illustrates an example process flow chart of a method, according to some implementations.

FIG. 11 illustrates a block diagram of an example computer system in which any of the implementations described herein may be implemented.

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide” includes a plurality of peptides.

1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein the following terms have the following meanings.

As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) claimed. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (−) 10%, 5% or 1%.

As used herein, the term “sequence identity” refers to a level of amino acid residue or nucleotide identity between two peptides or between two nucleic acid molecules. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A peptide (or a polypeptide or peptide region) has a certain percentage (for example, at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% or at least about 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. It is noted that, for any sequence (“reference sequence”) disclosed in this application, sequences having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% or at least about 99% sequence identity to the reference sequence are also within the disclosure.

Likewise, the present disclosure also includes sequences that have one, two, three, four, or five substitution, deletion or addition of amino acid residues or nucleotides as compared to the reference sequences.

In any of the embodiments described herein, analogs of a peptide comprising any amino acid sequence described herein are also provided, which have at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity to any of reference amino acid sequences. In some embodiments, the analogs include one, two, three, four, or five substitution, deletion or addition of amino acid residues as compared to the reference sequences. In some embodiments, the substitution is a conservative substitution.

As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. In some embodiments, non-natural amino acids are useful for tuning or engineering the helix or other secondary or tertiary structures of a peptide or protein for desired antimicrobial properties.

As is well-known in the art, a “conservative substitution” of an amino acid or a “conservative substitution variant” of a peptide refers to an amino acid substitution which maintains: 1) the secondary structure of the peptide; 2) the charge or hydrophobicity of the amino acid; and 3) the bulkiness of the side chain or any one or more of these characteristics. Illustratively, the well-known terminologies “hydrophilic residues” relate to serine or threonine. “Hydrophobic residues” refer to leucine, isoleucine, phenylalanine, valine or alanine, or the like. “Positively charged residues” relate to lysine, arginine, ornithine, or histidine. “Negatively charged residues” refer to aspartic acid or glutamic acid. Residues having “bulky side chains” refer to phenylalanine, tryptophan or tyrosine, or the like. A list of illustrative conservative amino acid substitutions is given in Table A.

TABLE A For Amino Acid Replace With Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D-Gln Aspartic Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser Glutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-Orn Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-Pro Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Alternatively, non-limiting examples of conservative amino acid substitutions are provided in Table B below, where a similarity score of 0 or higher indicates conservative substitution between the two amino acids.

TABLE B C G P S A T D E N Q H K R V M I L F Y W W −8 −7 −6 −2 −6 −5 −7 −7 −4 −5 −3 −3 2 −6 −4 −5 −2 0 0 17 Y 0 −5 −5 −3 −3 −3 −4 −4 −2 −4 0 −4 −5 −2 −2 −1 −1 7 10 F −4 −5 −5 −3 −4 −3 −6 −5 −4 −5 −2 −5 −4 −1 0 1 2 9 L −6 −4 −3 −3 −2 −2 −4 −3 −3 −2 −2 −3 −3 2 4 2 6 I −2 −3 −2 −1 −1 0 −2 −2 −2 −2 −2 −2 −2 4 2 5 M −5 −3 −2 −2 −1 −1 −3 −2 0 −1 −2 0 0 2 6 V −2 −1 −1 −1 0 0 −2 −2 −2 −2 −2 −2 −2 4 R −4 −3 0 0 −2 −1 −1 −1 0 1 2 3 6 K −5 −2 −1 0 −1 0 0 0 1 1 0 5 H −3 −2 0 −1 −1 −1 1 1 2 3 6 Q −5 −1 0 −1 0 −1 2 2 1 4 N −4 0 −1 1 0 0 2 1 2 E −5 0 −1 0 0 0 3 4 D −5 1 −1 0 0 0 4 T −2 0 0 1 1 3 A −2 1 1 1 2 S 0 1 1 1 P −3 −1 6 G −3 5 C 12

Alternatively, non-limiting examples of conservative amino acid substitutions include substitutions of a polar amino acid with a different polar amino acid, or substitutions of a hydrophobic amino acid with a different hydrophobic amino acid, as illustrated in Table C below. Each of the polar amino acids or hydrophobic amino acids, in some embodiments, can be substituted with Ala or Gly.

TABLE C Polar amino acids K, R, H, E, D, N, Q, S, T (or substituted with A or G) Hydrophobic amino V, I, L, M, C, F, W, Y acids (or substituted with A or G)

As used herein, the term “composition” refers to a preparation suitable for administration to an intended patient for therapeutic purposes that contains at least one pharmaceutically active ingredient, including any solid form thereof. The composition may include at least one pharmaceutically acceptable component to provide an improved formulation of the compound, such as a suitable carrier. In certain embodiments, the composition is formulated as a film, gel, patch, or liquid solution.

As used herein, the term “pharmaceutically acceptable” indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile.

2. Antimicrobial α-Helical Antimicrobial Peptides

The present disclosure, in some embodiments, describes a computational approach for generating a systemic formula from known α-helical antimicrobial peptides and using the formula to screen for new α-helical antimicrobial peptides. The systematic formula integrates features of idealized amphipathic and/or antimicrobial helices spanning up to 18 positions of canonical right-handed α-helices. Results demonstrate that nearly all families of known antimicrobial α-helical peptides align with the formula. In addition, many previously uncharacterized sequences were predicted to have direct antimicrobial activity. Synthesis of selected candidates and in vitro efficacy against human pathogens affirmed the veracity of model predictions and established validity of the α-core formula and search strategy. As a result, novel protein and peptide families and their specific sequences are identified as having potent and direct microbicidal efficacy that heretofore had not been ascribed.

The identified protein and peptide families and their specific sequences are provided in Tables 2-4 and SEQ ID NO:518-6860. In some embodiments, provided is an isolated peptide comprising an amino acid sequence of Table 2, 3 or 4 or any one of SEQ ID NO:518-6860, or an amino acid derived therefrom with one, two or three amino acid substitution. In some embodiments, the substitution is a conservative substitution. In some embodiments, the substitution is the replacement of a polar amino acid with a different polar amino acid (or A or G), or the replacement of a hydrophobic amino acid with a different hydrophobic amino acid (or A or G).

In some embodiments, provided is an isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 and an amino acid derived from anyone of SEQ ID NO: 1-14 with one, two or three amino acid substitution. In some embodiments, the substitution is a conservative substitution. In some embodiments, the substitution is the replacement of a polar amino acid with a different polar amino acid (or A or G), or the replacement of a hydrophobic amino acid with a different hydrophobic amino acid (or A or G). In some embodiments, the peptide comprises the amino acid sequence of anyone of SEQ ID NO: 1-14.

In some embodiments, the peptide is a fragment or fusion peptide described from natural proteins. In some embodiments, the peptide differs from natural proteins by at least an amino acid substation, addition or deletion.

In some embodiments, the peptide is not longer than 100 amino acid residues in length. In some embodiments, the peptide is not longer than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino acid resides in length. In some embodiments, the peptide has antimicrobial activity.

Fusion peptides are also described. In one embodiment, the present disclosure provides a fusion peptide comprising a first fragment selected from the sequences of Table 2, 3 or 4 or any one of SEQ ID NO:518-6860 or an amino acid derived from a sequence of Table 2, or 3 or any one of SEQ ID NO:518-6860 with one, or two or three amino acid substitution, and a second fragment having antimicrobial activity. In some embodiments, the second fragment comprises a gamma-core motif comprising two anti-parallel β-sheets interposed by a short turn region with a GXC or CXG sequence pattern integrated into one of the β-sheets.

As used herein, the terms “gamma-core motif,” or “γ-core,” and equivalents thereof refer to a multidimensional protein signature, in particular a multidimensional antimicrobial signature, that is characterized by two anti-parallel β-sheets interposed by a short turn region with a conserved GXC (dextromeric) or CXG (levomeric) sequence pattern integrated into one β-sheet. Additional features that characterize the y-core motif include a hydrophobic bias toward the C-terminal aspect and cationic charge positioned at the inflection point and termini of the β-sheet domains, polarizing charge along the longitudinal axis of the γ-core.

The kinocidin γ-core (γ_(KC) core) signature is an iteration of the antimicrobial peptide γ-core (γ_(AP)), conforming to an anti-parallel β-hairpin comprised of a 13-17 amino acid pattern with a central hydrophobic region typically flanked by basic residues. The γ_(KC) core motif can be characterized by the following consensus sequence formula:

NH₂[C]—[X₁₀₋₁₃]-[GX₂₋₃C]—[X₂]—[P]COOH

Human IL-8, which contains the kinocidin γ-core (γ_(KC) core) signature, has the sequence:

NH₂ CANTEIIVKLSDGRELCLDP COOH

This fragment of the IL-8 sequence is consistent with the consensus γ_(KC)-core motif. Furthermore, many kinocidins exhibit a recurring amino acid position pattern, consistent with the consensus γ_(KC) core formula:

where Z represents the hydrophobic residues A, F, I, L, V, W, Y; B represents the charged or polar residues D, E, H, K, N, R, Q; C, P, or G correspond to cysteine, proline, or glycine, respectively, X indicates variable amino acid position; and numeric superscripts of bracketed positions indicate relative frequency in percent, with common alternate residues listed beneath.

In one embodiment, the gamma-core motif comprises CPTAQLIATLKNGRKICLDLQ (SEQ ID NO: 15) or a first amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15. In one aspect, variants of the γ-core can include CPTAQLIATLKNGRKICLDLQP (SEQ ID NO: 16), CPTAQLIATLKNGRKICLDLQAP (SEQ ID NO: 17) and CPTAQLIATLKNGRKICLDLQA (SEQ ID NO: 18).

A linker can optionally be included between the first fragment and the second fragment, which is preferably 10 amino acids or fewer in length. In some aspect, the spacer is 9, 8, 6, 5, 4, 3, 2 amino acids in length or shorter. The spacer can include any amino acids, such as Ala, Pro, Cys, and Gly.

In some embodiments, the fusion peptide has antimicrobial activity. In some embodiments, the peptides may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

The peptides may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art. The peptides can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

3. Synthesis of Antimicrobial Peptides

The peptides described herein can be ordered from a commercial source or partially or fully synthesized using methods well known in the art (e.g., chemical and/or biotechnological methods). In certain embodiments, the peptides are synthesized according to solid phase peptide synthesis protocols that are well known in the art. In another embodiment, the peptide is synthesized on a solid support according to the well-known Fmoc protocol, cleaved from the support with trifluoroacetic acid and purified by chromatography according to methods known to persons skilled in the art. In other embodiments, the peptide is synthesized utilizing the methods of biotechnology that are well known to persons skilled in the art. In one embodiment, a DNA sequence that encodes the amino acid sequence information for the desired peptide is ligated by recombinant DNA techniques known to persons skilled in the art into an expression plasmid (for example, a plasmid that incorporates an affinity tag for affinity purification of the peptide), the plasmid is transfected into a host organism for expression, and the peptide is then isolated from the host organism or the growth medium, e.g., by affinity purification.

The peptides can be also prepared by using recombinant expression systems. Generally, this involves inserting the nucleic acid molecule into an expression system to which the molecule is heterologous (i.e., not normally present). One or more desired nucleic acid molecules encoding a peptide of the disclosure may be inserted into the vector. When multiple nucleic acid molecules are inserted, the multiple nucleic acid molecules may encode the same or different peptides. The heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′→3′) orientation relative to the promoter and any other 5′ regulatory molecules, and correct reading frame.

Purified peptides may be obtained by several methods. The peptide is preferably produced in purified form (preferably at least about 80% or 85% pure, more preferably at least about 90% or 95% pure) by conventional techniques. Depending on whether the recombinant host cell is made to secrete the peptide into growth medium (see U.S. Pat. No. 6,596,509 to Bauer et al., which is hereby incorporated by reference in its entirety), the peptide can be isolated and purified by centrifugation (to separate cellular components from supernatant containing the secreted peptide) followed by sequential ammonium sulfate precipitation of the supernatant. The fraction containing the peptide is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the peptides from other proteins. If necessary, the peptide fraction may be further purified by HPLC.

4. Antimicrobial Compositions and Formulations

Compositions and formulations that include any one or more of the peptides as disclosed herein are also provided. In one embodiment, the composition includes any one or more of the peptides and a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions of the disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They are preferably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

The pharmaceutical compositions of the disclosure can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. Formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.

Pharmaceutical formulations may be prepared as liquid suspensions or solutions using a sterile liquid, such as oil, water, alcohol, and combinations thereof. Pharmaceutically suitable surfactants, suspending agents or emulsifying agents, may be added for oral or parenteral administration. Suspensions may include oils, such as peanut oil, sesame oil, cottonseed oil, corn oil and olive oil. Suspension preparation may also contain esters of fatty acids, such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as poly(ethyleneglycol), petroleum hydrocarbons, such as mineral oil and petrolatum, and water may also be used in suspension formulations.

The compositions of this disclosure are formulated for pharmaceutical administration to a mammal, preferably a human being. Such pharmaceutical compositions of the disclosure may be administered in a variety of ways, preferably parenterally.

Sterile injectable forms of the compositions of this disclosure may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. Compounds may be formulated for parenteral administration by injection such as by bolus injection or continuous infusion. A unit dosage form for injection may be in ampoules or in multi-dose containers.

In addition to dosage forms described above, pharmaceutically acceptable excipients and carriers and dosage forms are generally known to those skilled in the art and are included in the disclosure. It should be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific peptide employed, the age, body weight, general health, sex and diet, renal and hepatic function of the patient, and the time of administration, rate of excretion, drug combination, judgment of the treating physician or veterinarian and severity of the particular disease being treated.

In some embodiments, the composition can further include a secondary antimicrobial agent. Non-limiting examples of such agents include imipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycin and daptomycin.

5. Therapeutic Methods

Methods of using the peptides, compositions and formulations of the present disclosure are also described. In one embodiment, the methods are for preventing or treating an infection of a microorganism. The microorganism can be a bacterium, such as a Gram-negative bacterium or a Gram-positive bacterium, a fungus, or a parasite.

The peptides, compositions and formulations are also useful for treating a disease or condition associated with an infection, such as wound abscess, catheter biofilm, pneumonia, and bacteremia.

In some embodiments, the treatment methods further include administration, concurrently or sequentially, of a second secondary antimicrobial agent. Non-limiting examples of such agents include imipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycin and daptomycin.

The peptides, compositions and formulations of the disclosure may be administered to the systemic circulation via parental administration. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. However, in cases where the infection is local (e.g., on the skin), the composition may be administered locally, such as topically.

6. Computational system and methods

The present disclosure, in some embodiments, provides computer-implemented methods for identifying antimicrobial sequences and related systems and non-transitory computer-readable media. In one embodiment, a computer-implemented method of identifying a peptide having antimicrobial activity is provided, as illustrated in FIG. 10 which is a process flow chart of a method 1000. The various processing operations and/or data flows depicted in FIG. 10 (and in the other drawing figures) are described in greater detail herein. The described operations may be accomplished using some or all of the system components described in detail above and, in some implementations, various operations may be performed in different sequences and various operations may be omitted. Additional operations may be performed along with some or all of the operations shown in the depicted flow diagrams. One or more operations may be performed simultaneously. Accordingly, the operations as illustrated (and described in greater detail below) are exemplary by nature and, as such, should not be viewed as limiting.

At step 1002, pursuant to a user command, the system, such as personal computing device, identifies a consensus formula from aligned amino acid sequences known to have an antimicrobial activity, such as those known as α-helical antimicrobial peptides. An example formula is X-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-X-[KRHEDNQSTAG]-[VILMCFWYAG], wherein X denotes any amino acid residue. The formula can be tested with a dataset that include known antimicrobial peptides and the formula can be further tuned (step 1004). For instance, the formula may be shortened or lengthened, or certain amino acid residues can include more or fewer substitutions.

The formula can then be used to search in a protein database (step 1006) for sequences or fragment that match the requirement of the formula. In some embodiments, the search query include one or more criteria such as location of the fragment in a protein, size of the protein, and organism of the protein (step 1008). In particular, in one embodiment, the search results are further evaluated for the presence of a signal peptide in the corresponding protein.

In some embodiments, the search results are further evaluated with respect to their biological, chemical, physical or sequential properties. Example properties include, without limitation, hydrophobic moment; mean hydrophobicity; net charge; frequencies or ratio of K and R; and isoelectric point (PI). Each of these evaluation scores can be used for prioritizing, ranking, filtering the search results (step 1012). Optionally, some of the search results are synthesized and tested in the lab for their antimicrobial activities.

FIG. 11 depicts a block diagram of an example computer system 1100 in which any of the embodiments described herein may be implemented. The computer system 1100 includes a bus 1102 or other communication mechanism for communicating information, one or more hardware processors 1104 coupled with bus 1102 for processing information. Hardware processor(s) 1104 may be, for example, one or more general purpose microprocessors.

The computer system 1100 also includes a main memory 1106, such as a random access memory (RAM), cache and/or other dynamic storage devices, coupled to bus 1102 for storing information and instructions to be executed by processor 1104. Main memory 1106 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 1104. Such instructions, when stored in storage media accessible to processor 1104, render computer system 1100 into a special-purpose machine that is customized to perform the operations specified in the instructions.

The computer system 1100 further includes a read only memory (ROM) 1108 or other static storage device coupled to bus 1102 for storing static information and instructions for processor 1104. A storage device 1110, such as a magnetic disk, optical disk, or USB thumb drive (Flash drive), etc., is provided and coupled to bus 1102 for storing information and instructions.

The computer system 1100 may be coupled via bus 1102 to a display 1112, such as a cathode ray tube (CRT) or LCD display (or touch screen), for displaying information to a computer user. An input device 1114, including alphanumeric and other keys, is coupled to bus 1102 for communicating information and command selections to processor 1104. Another type of user input device is cursor control 1116, such as a mouse, a trackball, or cursor direction keys for communicating direction information and command selections to processor 1104 and for controlling cursor movement on display 1112. This input device typically has two degrees of freedom in two axes, a first axis (e.g., x) and a second axis (e.g., y), that allows the device to specify positions in a plane. In some embodiments, the same direction information and command selections as cursor control may be implemented via receiving touches on a touch screen without a cursor.

The computing system 1100 may include a user interface module to implement a GUI that may be stored in a mass storage device as executable software codes that are executed by the computing device(s). This and other modules may include, by way of example, components, such as software components, object-oriented software components, class components and task components, processes, functions, attributes, procedures, subroutines, segments of program code, drivers, firmware, microcode, circuitry, data, databases, data structures, tables, arrays, and variables.

In general, the word “module,” as used herein, refers to logic embodied in hardware or firmware, or to a collection of software instructions, possibly having entry and exit points, written in a programming language, such as, for example, Java, C or C++. A software module may be compiled and linked into an executable program, installed in a dynamic link library, or may be written in an interpreted programming language such as, for example, BASIC, Perl, or Python. It will be appreciated that software modules may be callable from other modules or from themselves, and/or may be invoked in response to detected events or interrupts. Software modules configured for execution on computing devices may be provided on a computer readable medium, such as a compact disc, digital video disc, flash drive, magnetic disc, or any other tangible medium, or as a digital download (and may be originally stored in a compressed or installable format that requires installation, decompression or decryption prior to execution). Such software code may be stored, partially or fully, on a memory device of the executing computing device, for execution by the computing device. Software instructions may be embedded in firmware, such as an EPROM. It will be further appreciated that hardware modules may be comprised of connected logic units, such as gates and flip-flops, and/or may be comprised of programmable units, such as programmable gate arrays or processors. The modules or computing device functionality described herein are preferably implemented as software modules, but may be represented in hardware or firmware. Generally, the modules described herein refer to logical modules that may be combined with other modules or divided into sub-modules despite their physical organization or storage.

The computer system 1100 may implement the techniques described herein using customized hard-wired logic, one or more ASICs or FPGAs, firmware and/or program logic which in combination with the computer system causes or programs computer system 1100 to be a special-purpose machine. According to one embodiment, the techniques herein are performed by computer system 1100 in response to processor(s) 1104 executing one or more sequences of one or more instructions contained in main memory 1106. Such instructions may be read into main memory 1106 from another storage medium, such as storage device 1110. Execution of the sequences of instructions contained in main memory 1106 causes processor(s) 1104 to perform the process steps described herein. In alternative embodiments, hard-wired circuitry may be used in place of or in combination with software instructions.

The term “non-transitory media,” and similar terms, as used herein refers to any media that store data and/or instructions that cause a machine to operate in a specific fashion. Such non-transitory media may comprise non-volatile media and/or volatile media. Non-volatile media includes, for example, optical or magnetic disks, such as storage device 1110. Volatile media includes dynamic memory, such as main memory 1106. Common forms of non-transitory media include, for example, a floppy disk, a flexible disk, hard disk, solid state drive, magnetic tape, or any other magnetic data storage medium, a CD-ROM, any other optical data storage medium, any physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, NVRAM, any other memory chip or cartridge, and networked versions of the same.

Non-transitory media is distinct from but may be used in conjunction with transmission media. Transmission media participates in transferring information between non-transitory media. For example, transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise bus 1102. Transmission media can also take the form of acoustic or light waves, such as those generated during radio-wave and infra-red data communications.

Various forms of media may be involved in carrying one or more sequences of one or more instructions to processor 1104 for execution. For example, the instructions may initially be carried on a magnetic disk or solid state drive of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 1100 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector can receive the data carried in the infra-red signal and appropriate circuitry can place the data on bus 1102. Bus 1102 carries the data to main memory 1106, from which processor 1104 retrieves and executes the instructions. The instructions received by main memory 1106 may retrieves and executes the instructions. The instructions received by main memory 1106 may optionally be stored on storage device 1110 either before or after execution by processor 1104.

The computer system 1100 also includes a communication interface 1118 coupled to bus 1102. Communication interface 1118 provides a two-way data communication coupling to one or more network links that are connected to one or more local networks. For example, communication interface 1118 may be an integrated services digital network (ISDN) card, cable modem, satellite modem, or a modem to provide a data communication connection to a corresponding type of telephone line. As another example, communication interface 1118 may be a local area network (LAN) card to provide a data communication connection to a compatible LAN (or WAN component to communicated with a WAN). Wireless links may also be implemented. In any such implementation, communication interface 1118 sends and receives electrical, electromagnetic or optical signals that carry digital data streams representing various types of information.

A network link typically provides data communication through one or more networks to other data devices. For example, a network link may provide a connection through local network to a host computer or to data equipment operated by an Internet Service Provider (ISP). The ISP in turn provides data communication services through the world wide packet data communication network now commonly referred to as the “Internet”. Local network and Internet both use electrical, electromagnetic or optical signals that carry digital data streams. The signals through the various networks and the signals on network link and through communication interface 1118, which carry the digital data to and from computer system 1100, are example forms of transmission media.

The computer system 1100 can send messages and receive data, including program code, through the network(s), network link and communication interface 1118. In the Internet example, a server might transmit a requested code for an application program through the Internet, the ISP, the local network and the communication interface 1118.

The received code may be executed by processor 1104 as it is received, and/or stored in storage device 1110, or other non-volatile storage for later execution.

Each of the processes, methods, and algorithms described in the preceding sections may be embodied in, and fully or partially automated by, code modules executed by one or more computer systems or computer processors comprising computer hardware. The processes and algorithms may be implemented partially or wholly in application-specific circuitry.

The various features and processes described above may be used independently of one another, or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of this disclosure. In addition, certain method or process blocks may be omitted in some implementations. The methods and processes described herein are also not limited to any particular sequence, and the blocks or states relating thereto can be performed in other sequences that are appropriate. For example, described blocks or states may be performed in an order other than that specifically disclosed, or multiple blocks or states may be combined in a single block or state. The example blocks or states may be performed in serial, in parallel, or in some other manner. Blocks or states may be added to or removed from the disclosed example embodiments. The example systems and components described herein may be configured differently than described. For example, elements may be added to, removed from, or rearranged compared to the disclosed example embodiments.

Conditional language, such as, among others, “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and/or steps. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements and/or steps are included or are to be performed in any particular embodiment.

Any process descriptions, elements, or blocks in the flow diagrams described herein and/or depicted in the attached figures should be understood as potentially representing modules, segments, or portions of code which include one or more executable instructions for implementing specific logical functions or steps in the process. Alternate implementations are included within the scope of the embodiments described herein in which elements or functions may be deleted, executed out of order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved, as would be understood by those skilled in the art.

It should be emphasized that many variations and modifications may be made to the above-described embodiments, the elements of which are to be understood as being among other acceptable examples. All such modifications and variations are intended to be included herein within the scope of this disclosure. The foregoing description details certain embodiments of the invention. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways. As is also stated above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to including any specific characteristics of the features or aspects of the invention with which that terminology is associated. The scope of the invention should therefore be construed in accordance with the appended claims and any equivalents thereof.

Engines, Components, and Logic

Certain embodiments are described herein as including logic or a number of components, engines, or mechanisms. Engines may constitute either software engines (e.g., code embodied on a machine-readable medium) or hardware engines. A “hardware engine” is a tangible unit capable of performing certain operations and may be configured or arranged in a certain physical manner. In various example embodiments, one or more computer systems (e.g., a standalone computer system, a client computer system, or a server computer system) or one or more hardware engines of a computer system (e.g., a processor or a group of processors) may be configured by software (e.g., an application or application portion) as a hardware engine that operates to perform certain operations as described herein.

In some embodiments, a hardware engine may be implemented mechanically, electronically, or any suitable combination thereof. For example, a hardware engine may include dedicated circuitry or logic that is permanently configured to perform certain operations. For example, a hardware engine may be a special-purpose processor, such as a Field-Programmable Gate Array (FPGA) or an Application Specific Integrated Circuit (ASIC). A hardware engine may also include programmable logic or circuitry that is temporarily configured by software to perform certain operations. For example, a hardware engine may include software executed by a general-purpose processor or other programmable processor. Once configured by such software, hardware engines become specific machines (or specific components of a machine) uniquely tailored to perform the configured functions and are no longer general-purpose processors. It will be appreciated that the decision to implement a hardware engine mechanically, in dedicated and permanently configured circuitry, or in temporarily configured circuitry (e.g., configured by software) may be driven by cost and time considerations.

Accordingly, the phrase “hardware engine” should be understood to encompass a tangible entity, be that an entity that is physically constructed, permanently configured (e.g., hardwired), or temporarily configured (e.g., programmed) to operate in a certain manner or to perform certain operations described herein. As used herein, “hardware-implemented engine” refers to a hardware engine. Considering embodiments in which hardware engines are temporarily configured (e.g., programmed), each of the hardware engines need not be configured or instantiated at any one instance in time. For example, where a hardware engine comprises a general-purpose processor configured by software to become a special-purpose processor, the general-purpose processor may be configured as respectively different special-purpose processors (e.g., comprising different hardware engines) at different times. Software accordingly configures a particular processor or processors, for example, to constitute a particular hardware engine at one instance of time and to constitute a different hardware engine at a different instance of time.

Hardware engines can provide information to, and receive information from, other hardware engines. Accordingly, the described hardware engines may be regarded as being communicatively coupled. Where multiple hardware engines exist contemporaneously, communications may be achieved through signal transmission (e.g., over appropriate circuits and buses) between or among two or more of the hardware engines. In embodiments in which multiple hardware engines are configured or instantiated at different times, communications between such hardware engines may be achieved, for example, through the storage and retrieval of information in memory structures to which the multiple hardware engines have access. For example, one hardware engine may perform an operation and store the output of that operation in a memory device to which it is communicatively coupled. A further hardware engine may then, at a later time, access the memory device to retrieve and process the stored output. Hardware engines may also initiate communications with input or output devices, and can operate on a resource (e.g., a collection of information).

The various operations of example methods described herein may be performed, at least partially, by one or more processors that are temporarily configured (e.g., by software) or permanently configured to perform the relevant operations. Whether temporarily or permanently configured, such processors may constitute processor-implemented engines that operate to perform one or more operations or functions described herein. As used herein, “processor-implemented engine” refers to a hardware engine implemented using one or more processors.

Similarly, the methods described herein may be at least partially processor-implemented, with a particular processor or processors being an example of hardware. For example, at least some of the operations of a method may be performed by one or more processors or processor-implemented engines. Moreover, the one or more processors may also operate to support performance of the relevant operations in a “cloud computing” environment or as a “software as a service” (SaaS). For example, at least some of the operations may be performed by a group of computers (as examples of machines including processors), with these operations being accessible via a network (e.g., the Internet) and via one or more appropriate interfaces (e.g., an Application Program Interface (API)).

The performance of certain of the operations may be distributed among the processors, not only residing within a single machine, but deployed across a number of machines. In some example embodiments, the processors or processor-implemented engines may be located in a single geographic location (e.g., within a home environment, an office environment, or a server farm). In other example embodiments, the processors or processor-implemented engines may be distributed across a number of geographic locations.

Language

Throughout this specification, plural instances may implement components, operations, or structures described as a single instance. Although individual operations of one or more methods are illustrated and described as separate operations, one or more of the individual operations may be performed concurrently, and nothing requires that the operations be performed in the order illustrated. Structures and functionality presented as separate components in example configurations may be implemented as a combined structure or component. Similarly, structures and functionality presented as a single component may be implemented as separate components. These and other variations, modifications, additions, and improvements fall within the scope of the subject matter herein.

Although an overview of the subject matter has been described with reference to specific example embodiments, various modifications and changes may be made to these embodiments without departing from the broader scope of embodiments of the present disclosure. Such embodiments of the subject matter may be referred to herein, individually or collectively, by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single disclosure or concept if more than one is, in fact, disclosed.

The embodiments illustrated herein are described in sufficient detail to enable those skilled in the art to practice the teachings disclosed. Other embodiments may be used and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. The Detailed Description, therefore, is not to be taken in a limiting sense, and the scope of various embodiments is defined only by the appended claims, along with the full range of equivalents to which such claims are entitled.

It will be appreciated that an “engine,” “system,” “data store,” and/or “database” may comprise software, hardware, firmware, and/or circuitry. In one example, one or more software programs comprising instructions capable of being executable by a processor may perform one or more of the functions of the engines, data stores, databases, or systems described herein. In another example, circuitry may perform the same or similar functions. Alternative embodiments may comprise more, less, or functionally equivalent engines, systems, data stores, or databases, and still be within the scope of present embodiments. For example, the functionality of the various systems, engines, data stores, and/or databases may be combined or divided differently.

“Open source” software is defined herein to be source code that allows distribution as source code as well as compiled form, with a well-publicized and indexed means of obtaining the source, optionally with a license that allows modifications and derived works.

The data stores described herein may be any suitable structure (e.g., an active database, a relational database, a self-referential database, a table, a matrix, an array, a flat file, a documented-oriented storage system, a non-relational No-SQL system, and the like), and may be cloud-based or otherwise.

EXAMPLES Example 1. Identification of New Antimicrobial α-Core Helices

This example describes an experiment in which a consensus sequence formula was developed and used to search for new α-helical antimicrobial peptides (AHAPs) having microbiocidal activities.

Among the most potent natural antibiotics known to date include α-helical host defense peptides. These peptides effect a first line of defense against invading pathogens and have been isolated from species ranging from microbes to man. While many prior investigations have analyzed individual and class-specific properties through which these peptides convey function, machine-learning strategies to define unifying principles and underlying structure activity relationships (SARs) have been limited. In this example, a systematic formula encompassing features of idealized amphipathic and/or antimicrobial helices that span up to 18 positions of canonical right-handed α-helices was designed. The formula was then applied to search known protein databases seeking known as well as unforeseen proteins or peptides fulfilling its structural signature. Results demonstrate that nearly all families of known antimicrobial α-helical peptides align with this formula. Interestingly, logical search algorithms using this formula discovered many previously uncharacterized sequences predicted to have direct antimicrobial activity. Laboratory studies affirmed the veracity of predictions and established validity of the α-core formula and search strategy. As a result, new protein and peptide families and specific sequences are identified as having potent and direct microbicidal efficacy that heretofore had not been ascribed.

Methods and Materials Identification of the α-Core Formula

To identify a consensus formula that was representative of nearly all classes of AHAPs, multiple sequence alignments with prototypical α-helical peptides using CLUSTAL W (www.ebi.ac.uk/Tools/msa/clustalw2/) were carried out. Alignments were then manually adjusted at certain positions using MEGA 6. Through iterative refinements of this process, an 18 residue generalized amphipathic formula emerged with sequence degeneracies at each position that was representative of nearly all classes of AHAPs. This formula can initiate at any of the positions that make up a standardized 18 residue α-helical wheel.

Assignment of Residue Polarity within the α-Core Formula

Within the formula, individual residues were categorized as either hydrophobic or hydrophilic as per the Wimley-White hydrophobicity scale, a scale that has been empirically derived and includes contributions from the peptide bond. One exception was for alanine (A), which was also included with the hydrophobic residues as per the Eisenberg and Kyte-Doolittle hydrophobicity scales. This assignment was made in-part due to preliminary studies that frequently localized alanine to the hydrophobic facet of many antimicrobial peptides.

Accuracy of Formula in Retrieving Helical Sequences

The amphipathic helical consensus formula above was queried against the PDB 3D database (www.wwpdb.org) to assess the fidelity of the formula in identifying helical domains. The first 100 non-redundant retrieved structures were scored for helicity of the target sequence. Proteins were considered to be a positive hit if the target sequence was more than 75% helical.

Use of α-Core Formula as a Database Query

The α-core sequence formula was used with the ScanProsite (prosite.expasy.org/scanprosite/) tool to carry out iterative pattern searches of the UniProtKB Swiss-Prot database. While initial searches queried the database with varying lengths of the amphipathic sequence formula, it was ultimately found that a relatively short query sequence of 12 residues was most efficient at retrieving the majority of antimicrobial peptide sequences. Iteration 1 of this query sequence is listed below:

X-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]- [VILMCFWYAG]-[VILMCFWYAG]-[KRHEDNQSTAG]- [KRHEDNQSTAG]-[VILMCFWYAG]-X-[KRHEDNQSTAG]- [VILMCFWYAG]

After this optimization process, the sequence formula was used as a query against the UniProtKB Swiss-Prot and TrEMBL databases. The formula was advanced one position at a time through 18 iterations to represent an entire 18-residue helical wheel span. ScanProsite search results were further limited by: 1) protein size (<200 residues); 2) eukaryotic organisms; and 3) localization of the pattern to the C-terminal region using a “X(0,50)>” logical operator.

Signal Peptide and Biophysical Parameter Determination

Retrieved datasets were additionally screened for the presence of a signal peptide using SignalP 4.1 (www.cbs.dtu.dk/services/SignalP/). Hydrophobic moment (μH), mean hydrophobicity (H), net charge (Q−K and R (+1); H (+0.5); D and E (−1)) and K and R residue frequency were determined in batch using Python algorithms created for this purpose. PI was determined using the ExPasy Compute PI tool (web.expasy.org/compute_pi/).

Candidate Peptides and HDPs

Select candidate microbicidal peptides were commercially synthesized by BioMatik (Biomatik USA, Wilmington, Del.). Lyophilized peptides were reconstituted with ddIH20 and stored in aliquots at −20° C. LL-37 (Peptides International, Louisville, Ky.), a prototypic human AHAP, was used as a comparator in microbicidal assays.

Assay for Antimicrobial Activity

Putative antimicrobial peptides were assayed for microbicidal activity using a well-established radial diffusion method modified to pH 5.5 or 7.5. A panel of microorganisms was tested: Gram-positive Staphylococcus aureus (ISP 479C, ISP 479R); Gram-negative Salmonella typhimurium (MS 5996s, MS 14028), Pseudomonas aeruginosa (PA01), Acinetobacter baumanni (19606) and the fungus Candida albicans (36082S, 36082R). Logarithmic phase organisms were inoculated (10⁶ CFU/ml) into buffered agarose, and poured into plates. Peptides (10 μg) were introduced into wells in the seeded matrix, and incubated for 3 h at 37° C. Nutrient overlay medium was applied, and assays incubated at 37° C. or 30° C. for bacteria or fungi, respectively. After 24 h, zones of inhibition were measured. Independent experiments were repeated a minimum of two times.

Results Derivation and Iterative Refinement of the α-Core Sequence Formula

In initial studies, alignments with prototypic representatives from all of the major classes of AHAPs were carried out to identify conserved sequence elements. This analysis revealed that nearly all of these prototypes could be aligned with a degenerate sequence formula wherein either polar or non-polar residues were assigned to positions along an amphipathic helix. Peptides adhered to this formula ranging from a minimal 11 to maximal 16 residue span corresponding to 3 to 4.5 turns of an α-helix. Based on this analysis, a consensus formula representative of nearly all of the major classes of AHAPs was extracted (FIGS. 1A-B and 2).

Once this preliminary consensus was defined, it was further refined by testing its efficiency to return α-helical domains when used as a query against the PDB protein structure database. As a baseline, versions of the formula lacking known helix breakers proline and glycine were carried out. Results of these analyses revealed that the efficiency of the formula in retrieving α-helical domains was very high, identifying these motifs (defined as spans that were at least 75% helical) with a frequency of 92-94%.

As many AHAPs are known to contain proline and glycine residues, subsequent tests to evaluate the impact of these residues on the efficiency of the formula were carried out. When proline was included within the sequence formula, α-helical domains within target sequences were retrieved less than 10% of the time. These findings strongly support the theory that proline, due to its side chain and steric constraints, is inconsistent with stable α-helix formation. Because of this, proline was excluded from the formula.

In contrast when glycine was allowed within the sequence formula, queries against the structure database revealed two different outcomes depending on whether targets had been determined in aqueous or non-polar environments. For structures determined in aqueous environments, the sequence formula retrieved α-helical domains approximately 74% of the time when a single glycine was present; a value that was reduced to 40% when two or more glycine residues were present. By comparison, when target structures were determined in non-polar environments (high levels of TFE, SDS, or micellar components), the presence of glycine had very little impact on peptide helicity. These studies revealed that the sequence formula retrieved α-helical domains approximately 92% if the time when as many as four glycine residues were present in the target sequence. Because of this, and since glycine is highly represented in AHAPs, this residue was included in the sequence formula. Moreover, a large number of biophysical studies have demonstrated that while many glycine-rich AHAPs are unstructured in aqueous environments, they nearly always adopt an α-helical conformation when interacting with the hydrophobic membrane environments of their microbial targets.

Alanine/Glycine Both Faces of Helix

While the assignment of most residues to either the polar or non-polar residue group within the sequence formula was relatively straightforward based on the initial AHAP consensus and known residue-specific hydrophobicity values, the placement of alanine and glycine was less obvious due to their relatively neutral AG values in polar and non-polar environments. Because of this, the requirement for glycine and/or alanine on either the polar or non-polar face of the helix was assessed in an iterative manner via queries against a control dataset comprised of more than 400 AHAP peptides (FIG. 3). Preliminary versions of the formula, lacking alanine and glycine, failed to retrieve significant numbers (>10%) of the control AHAP study set (FIG. 3). The inclusion of alanine on the polar face of the amphipathic helix increased the retrieved fraction of peptides to 26%, and when alanine was added to both the polar and non-polar search terms the retrieved AHAPs increased to 48%. With respect to glycine a similar relationship was found. The addition of glycine to the polar face of the query sequence led to the identification of approximately 81% of the control AHAP dataset, and when glycine was included on both the polar and non-polar facets, the query retrieved close to 99% of the AHAP study set. As a result of this analysis, alanine and glycine were included as components of both the polar and non-polar query search terms.

α-Core Sequence Formula

Based on the above considerations, a linear α-core formula spanning all 18 positions of a canonical right-handed alpha helix was created, as shown below. When translated into three dimensions, the formula describes an idealized amphipathic helix, with distinct hydrophobic and hydrophilic facets. Positions in between the polar and nonpolar faces were assigned a value of “X” within the linear formula, leading to a polar angle (θ) maxima of 180° and minima of 140° . Due to the degenerate nature of the formula, each position along the helical span was represented by multiple polar or non-polar residues (below and FIGS. 1A-B and 2).

Pos_((n 0-17)) = [(sin  n δ), (cos  n δ)] $\delta = {100\mspace{14mu} {for}\mspace{14mu} {standard}\mspace{14mu} \alpha \text{-}{{helix}\begin{bmatrix} {1\mspace{14mu} 2\mspace{14mu} 3\mspace{14mu} 4\mspace{14mu} 5\mspace{14mu} 6\mspace{14mu} 7\mspace{14mu} 8\mspace{14mu} 9\mspace{14mu} 10\mspace{14mu} 11\mspace{14mu} 12\mspace{14mu} 13\mspace{14mu} 14\mspace{14mu} 15\mspace{14mu} 16\mspace{14mu} 17\mspace{14mu} 18} \\ {X\text{-}\left( {P\text{-}H\text{-}H\text{-}P\text{-}P\text{-}H\text{-}H\text{-}P} \right)\text{-}X\text{-}\left( {H\text{-}P\text{-}P\text{-}H\text{-}H\text{-}P\text{-}P\text{-}H} \right)} \end{bmatrix}}_{n = {0\text{-}18}}}$ P(polar) = [EDKRHQNTSAG] H(hydrophobic) = [VMCILFWYAG]

Primary Database Searches Using the α-Core Sequence Formula

Once refined, the α-core sequence formula was used as a query against the SwissProt and TrEMBL protein sequence databases (FIG. 4). As described in Methods, database queries were further limited to sequences that were eukaryotic in origin, less than 200 residues in length and within 50 residues of the C-terminus.

Returned raw datasets consisted of more than 70,000 sequences, often representing multiple target hits shifted along amphipathic helical spans of a single protein. Each of these returned hits was scored for hydrophobic moment, and sequences with the highest pH were extracted to generate a non-redundant dataset of approximately 13,000 unique sequences for downstream studies. Dataset proteins were also scored for the presence of a signal peptide, and sequences lacking this motif were removed to generate a final dataset of approximately 3,800 sequences.

Efficiency of the α-Core Sequence Formula

The results of the above database queries indicated that the amphipathic sequence algorithm was highly robust, retrieving members from nearly all of the known AHAP families (Table 1). Overall, the formula retrieved at least one member of more than 106 different helical peptide families representing approximately 94%, of all known AHAP classifications. Moreover, while the formula describes an idealized amphipathic helical structure, it nonetheless returned 827 individual antimicrobial peptide sequences, representing approximately 88% of all known AHAPs in the SwissProt database.

TABLE 1 α-Helical Peptide Families Retrieved with Alpha-Core Formula Search Class Peptide Organism 1 Cnidaria Clavanins Sea squirt 2 Clavaspirin Sea squirt 3 Halocidin Sea peach 4 Halocyntin Sea squirt 5 Styelins Sea squirt 6 Plants Ginkbilobin Ginko 7 Thionin (helical domain) Wheat 8 Arthropods Amphipathic peptides Scorpion 9 Andropin Fruit fly 10 Anionic Antimicrobial peptide 2 Moth 11 Antifungal Protein MAF-1 Fly 12 Antimicrobial peptides (36.4, 143) Scorpion 13 Antimicrobial peptide ctriporin Scorpion 14 Antimicrobial peptide HsAp1 Scorpion 15 Bactericidin Moth 16 Bombolitin Bee 17 Cecropins Moth/fly 18 Cecropin-D-like peptide Moth 19 Cryptonin Cicada 20 Cytotoxic linear peptide Scorpion 21 Decoralin Wasp 22 Dominulins Wasp 23 Eumenitins Wasp 24 Hadrurin Scorpion 25 Im-1 Scorpion 26 Imcroporin Scorpion 27 Lebocin-like peptide Moth 28 Mastoparans Wasp 29 Mastoparan-like peptides Wasp 30 Melittin Bee 31 Meucin-49 Scorpion 32 Moricins Moth 33 Moricin-like peptide A Moth 34 Mucroporin Scorpion 35 Mucroporin-like peptide Scorpion 36 Non-disulfide-bridged peptides Scorpion 37 Oxyopinin 4a Spider 38 Pandinin Scorpion 39 Parabutoporin Scorpion 40 Peptide BmKb1 Scorpion 41 Peptides Ctry Scorpion 42 Peptides Hp (Non-disulfide bridged) Scorpion 43 Pilosulins Ant 44 Ponericins Ant 45 Ponericin W-like Scorpion 46 Protonectin Wasp 48 Stomoxyn fly 49 Fish Chrysophsin Sea Bream 50 Pleurocidin Flounder 51 Pleurocidin-like peptide Flounder 52 Grammistins Soapfish 53 Pardaxins Sole 54 Piscidin 3 Striped bass 55 Amphibians Ascaphin Frog 56 Aureins Frog 57 Antimicrobial peptide PGQ Frog 58 Antimicrobial peptides Frog 59 Brevinins Frog 60 Bombesin Toad 61 Bombinins Toad 62 Bombinin-like peptides Toad 63 Caeridin Frog 64 Caerins Frog 65 Citropins Frog 66 Cyanophlyctin Frog 67 Dahleins Frog 68 Dermadistinctins Frog 69 Dermaseptins Frog 70 Dermaseptin-like peptides Frog 71 Dermatoxin Frog 72 Distinctin-like peptide Frog 73 Esculentins Frog 74 Fallaxidins Frog 75 Frenatins Frog 76 Gaegurin Frog 77 Grahamins Frog 78 Guentherin Frog 79 Hylins Frog 80 Maximins Toad 81 Nigrocins Frog 82 Ocellatins Frog 83 Palustrins Frog 84 Phylloseptins Frog 85 Pseudins Frog 86 Prolevitide Frog 87 Ranatuerins Frog 88 Rugosins Frog 89 Syphaxin Frog 90 Temporins Frog 91 Xenopsin Frog 92 Uperins Frog 93 Reptiles CRAMPs Snake 94 Birds Cathelicidins Chicken 95 Mammals Cathelicidins Mammals 96 Cathelicidin related peptide SC5 Sheep 97 CRAMP Mouse 98 CAP-18 Rabbit 99 PMAP-37 Pig 100 Chemokines (helical domain) Mammals 101 CXCL 102 CCL 103 XCL 104 Granulysin Human 105 Dermcidin Human 106 NK-lysin Horse

Beyond retrieving a high proportion of known AHAPs, when limited to sequences with a PI of 8.5 or greater, the formula was also relatively specific in returning these sequences. Searches were carried out in several stages of either 0-50, or 51-200 residues, depending on whether a signal peptide test was applied. For the 0-50 residue set, approximately 71% of the retrieved sequences were known AHAPs; whereas, for the 51-200 residue set, approximately 27% of the identified sequences were α-helical antimicrobial proteins. Given that there are approximately 940 (see methods) named AHAPs in the 553,000 sequence SwissProt database (version 12-1-16), this represents and in-silico enrichment of approximately 130-fold.

With respect to other proteins retrieved by the sequence formula, the second most highly represented group were a variety of toxin peptides making up approximately 17% of the dataset. Beyond this, other protein families that were abundant in the retrieved dataset included: apovitellins, leptins, vasoactive intestinal proteins and uncharacterized proteins.

Residue Frequency

Given that the α-core sequence formula returns aligned datasets, it was possible to score identified AHAP helices for the abundance of various residues at each position along an 18 residue amphipathic span. Moreover, given that the α-core formula identifies amphipathic patterns that are likely to form helices, it was also possible to make assumptions about the localization of specific resides to either the polar or non-polar facet of these structures. Due to the significant evolutionary distance between invertebrate and lower and higher vertebrate classes of AHAPs, the dataset was divided into three groups representing arthropod, amphibian and mammalian sequences (FIG. 5).

The analysis of residue frequency revealed a number of findings regarding the composition and hydrophilic and hydrophobic distribution of residues within returned AHAP helices. One finding of note was that glycine was highly represented in AHAPs of lower organisms, comprising approximately 30-35% of all residues in target amphipathic spans in arthropods and amphibians. While glycine was still abundant in mammals, it occurred less frequently representing about 15% of residues in the returned sequences.

It was also of interest to find that alanine, while abundant in amphipathic spans of arthropods and amphibians, was much less common in the identified sequences of mammals. Moreover, while alanine was found more frequently on the hydrophobic face of the peptide, it was also found with some frequency 10-30% on the polar face of peptides from arthropods and amphibians.

With respect to charged residues, it is of interest that lysine was the most abundant polar residue in non-mammalian species (30%) and was strongly preferred over arginine at an approximately 12:1 ratio. By contrast, in mammals this preference was markedly reduced, with this ratio decreasing to a 3:1 abundance of lysine to arginine. It is also of note to find negatively charged aspartic and glutamic acid residues within the returned sequences at a low frequency of approximately 5% in all species groups.

Biophysical Signatures

Beyond measuring the frequency of specific residues within returned AHAP helices, sequences were also scored for a number of biophysical parameters including: net charge (Q), isoelectric point (PI), hydrophobic moment (μH), hydrophobicity (H), and lysine to arginine (K/K+R) ratio.

In this analysis it was found that the average net charge for the AHAP subset of study helices (n=803) was +2.0. If only cationic sequences (Q≥0.5; n=707) were considered this value rises to +2.7. By comparison the mean net charge for study toxin helices (n=717) was +1.3, and +0.6 for other study helices. With respect to hydrophobic moment, AHAP helices had an average μH of 0.52 while toxins had an average pH 0.42 and other peptides 0.39. While some reports suggest that mean hydrophobicity may be greater for toxins than for AHAPs, in this study values for hydrophobicity in the retrieved helices were similar, with an average H of 0.42 for AHAPs, and 0.34 for toxins as compared with other peptides (0.39).

Negative Gaussian Curvature

Retrieved sequences were also scored for their ratio of lysine to arginine (N_(K)/N_(K)+N_(R)) residues. Prior studies have demonstrated that the relative abundance of these residues can significantly impact the propensity of a given peptide to induce negative Gaussian curvature (NGC) in membranes, a phenomenon that is associated with pore formation and membrane permeabilization. Prior studies have demonstrated that the residue lysine is favored over arginine in AHAPs, a substitution that necessitates an increased level of peptide hydrophobicity to efficiently induce NGC. In the present example, retrieved AHAP helices largely adhered to this rule, demonstrating a modest positive association between N_(K)/N_(K)+N_(R) ratio and peptide hydrophobicity (FIG. 6). By comparison, amphipathic helices from toxins did not comply with this observation, where a negative relationship between N_(K)/N_(K)+N_(R) ratio and peptide hydrophobicity was found.

Application of the α-Core Sequence Formula to Retrieve Previously Uncharacterized Antimicrobial Sequences

In addition to the known classes of antimicrobial proteins, the α-core formula also retrieved many uncharacterized proteins, or proteins with an alternate primary function. Based on the efficiency with which the formula retrieved antimicrobial peptides, it was of interest to determine whether some of these additional sequences might represent: 1) as yet uncharacterized antimicrobial proteins; or 2) proteins with an assigned function that also have an as yet unidentified antimicrobial function or antimicrobial domain within a larger protein.

To identify putative microbicidal sequences, study set peptides were scored for the biophysical parameters described above (μH, Q, H, K/K+R, PI) and analyzed to determine which factors and/or relationships were most efficient at separating known AHAPs from non-microbicidal sequences. Results from these analyses, demonstrated that mean amphipathicity (μH) and either net charge (Q) (FIG. 7) or PI were the most efficient means of discriminating bona-fide AHAPs from other sequences.

Based on the above analyses, dataset proteins were scored for either cationicity or PI and hydrophobic moment (Q*μH) or (PI*μH) and sequences of interest were prioritized. While this analysis retrieved many peptides of interest, several families of biological significance were consistently found amongst the top scoring sequences. These included members of the γ-chain-dependent interleukin family along with many interferon sequences. Moreover, a number of consistently high scoring peptides were also derived from the globally-impactful plant pathogen Phytophthora parasitica.

Given the above considerations, ten lead candidates representing these families were synthesized so that their antimicrobial properties could be determined (Table 2). As shown in Table 2, they included:

Interleukins

IL-5—Meleagris gallopavo (common turkey)

IL-7—human

IL-13—human

IL-21—human

Interferons

IFN-γ—Myotis davidii (vesper bat)

IFN-γ—human

Phytophthora parasitica Uncharacterized Sequences

Pp-1—P. parasitica

Pp-2—P. parasitica

Pp-3—P. parasitica

Pp-4—P. parasitica

Two additional candidates were identified when the localization of the pattern relative to the C-terminal region was relaxed. These two candidates, along with two respective longer versions are listed in Table 3. Additional listings of identified peptides are provided in Table 4, and SEQ ID NO:518-6029 (localization required) and SEQ ID NO:6030-6860 (localization requirement relaxed).

TABLE 2 Study set peptides SEQ Short ID Name Peptide Accession Species Sequence Charge μH NO: tIL-5 IL-5 G1N9U6 Meleagris KRFIEKLRTF LRKLSRGAK +7 0.68  1 gallopavo hIL7 IL-7 P13232 Homo sapiens LCFLKRLLQE IKTCWNKILM +3.5 0.47  2 GTKEH hIL-13 IL-13 P35225 Homo sapiens EVAQFVKDLL LHLKKLFREG +2.5 0.39  3 RFN hIL-21 IL-21 Q9HBE4 Homo sapiens KEFLERFKSL LQKMIHQHLS +2.5 0.32  4 SRTHGSEDS bIFN-γ IFN-g L5LME8 Myotis KAISELYNVI TELSKSNSKM +8 0.40  5 davidii RKRRQNLFRG WKASK hIFN-γ IFN-g P01579 Homo sapiens AIHELIQVMA ELSPAAKTGK +6 0.23  6 RKRSQMLFRG RRASQ Pp-1 W2QY53 Phytophthora TQAWTNFKKW FKKWFKKTFS +6 0.59  7 parasitica Pp-2 W2N8A5 Phytophthora LFQKIKRWWK RIFEHEASRS +7.5 0.47  8 parasitica ARRLRAV Pp-3 W2HTE6 Phytophthora KEVIKKFTKA MEKMKKNGRA +6 0.54  9 parasitica Pp-4 A0A080YYV3 Phytophthora  LWQRFLRWWN RLFHVSSNRL +6.5 0.42 10 parasitica LREGGKK

TABLE 3 Additional peptides identified with relaxed  localization requirement SEQ Acces- ID Peptide sion Species Sequence Length Charge μH NO Dynorphin P01213 Homo YGGFLRRIRP KLKWDNQ 17 +4 0.32 11 A (short) sapiens Dynorphin P01213 Homo YGGFLRRIRP KLKWDNQKRY 32 +9 0.16 13 (long) sapiens GGFLRRQFKV VT Oncostatin P13725 Homo KEFLERFKSL LQKMIHQHLS 29 +2.5 0.32 12 M (short) sapiens SRTHGSEDS Oncostatin P13725 Homo RFLHGYHRFM HSVGRVFSKW 60 +19.5 0.32 14 M (long) sapiens GESPNRSRRH SPHQALRKGV RRTRPSRKGK RLMTRGQLPR

TABLE 4 Priority alpha core peptide sequences SEQ Acces- ID sion # Name Comp Form Match HM Q HM*Q PI NO: W7G0H8 Uncharacterized protein RIKKFLKRCCKKIK 0.82 8 6.52 6.17  19 W4IIC4 Uncharacterized protein RIKKFLKRCCKKIK 0.82 8 6.52 6.03  20 A0A024W805 Uncharacterized protein RIKKFLKRCCKKIK 0.82 8 6.52 6.03  21 U3KN10 Uncharacterized protein FLKKLLQKIKTCWNKILR 0.75 6 4.51 8.7  22 GIKE A0A016VH75 Uncharacterized protein KRVKRFCKKACRKASQ 0.56 8 4.47 9.2  23 A0A016VGG2 Uncharacterized protein KRVKRFCKKACRKASQ 0.56 8 4.47 9.2  24 G1N9U6 Uncharacterized protein KNVKRFIEKLRTFLRKLS 0.72 6 4.34 9.44  25 Q8SUJ0 Uncharacterized protein LVRKIIKYCRKL 0.86 5 4.28 5.53  26 M1JIG5 Uncharacterized protein LVRKIIKYCRKL 0.86 5 4.28 5.53  27 T1HZ04 Uncharacterized protein FRRLCRNIKEVIKK 0.85 5 4.25 6.15  28 A0A080YVY9 Uncharacterized protein TQAWTNFKKWFKKWFKK 0.69 6 4.13 9.23  29 W2QY53 Uncharacterized protein TQAWTNFKKWFKKWFKK 0.69 6 4.13 9.23  30 W2W791 Uncharacterized protein TQAWTNFKKWFKKWFKK 0.69 6 4.13 9.23  31 W2G110 Uncharacterized protein TQAWTNFKKWFKKWFKK 0.69 6 4.13 9.23  32 E0AD11 Interleukin-5 VKKFIEKLRTFIRKL 0.82 5 4.08 9.06  33 Q5W4T8 Interleukin-5 VKKFIEKLRTFIRKL 0.82 5 4.08 8.88  34 F1P2P6 Uncharacterized protein VKKFIEKLRTFIRKL 0.82 5 4.08 8.88  35 A0A093FG05 Interleukin-21 (Fragment) KEFLKSFAKLIRKVIR 0.80 5 4.02 9.54  36 A0A094LKV9 Interleukin-21 (Fragment) KEFLKSFAKLIKKVIR 0.80 5 4.01 9.57  37 A0A091RQW4 Interleukin-21 (Fragment) KEFLKSFAKLIKKVIR 0.80 5 4.01 9.49  38 A0A091U0R6 Interleukin-21 (Fragment) KEFLKSFAKLIKKVIR 0.80 5 4.01 9.44  39 A0A091WDE2 Interleukin-21 (Fragment) KEFLKSFAKLIKKVIR 0.80 5 4.01 9.37  40 A0A091Q048 Interleukin-21 (Fragment) KEFLKSLAKLIKKVIR 0.80 5 3.99 9.61  41 Q32620 Uncharacterized 3.3 kDa protein  FFKWISKFIRRLSKCG 0.79 5 3.95 10.74  42 in psbT-psbN intergenic region (ORF27) H2WTG9 Uncharacterized protein ANKANRMMRKIMRKL 0.66 6 3.94 4.9  43 F6W4B4 Uncharacterized protein (Fragment) WYQLIRTFGNLIHQKYRKL 0.59 6.5 3.86 9.3  44 LEAYRKLR P56478 Interleukin-7 (IL-7) FLKRLLREIKTCWNKILKG 0.77 5 3.83 8.91  45 Q544C8 Interleukin 7 (Interleukin 7, FLKRLLREIKTCWNKILKG 0.77 5 3.83 8.73  46 isoform CRA_b) P10168 Interleukin-7 (IL-7) FLKRLLREIKTCWNKILKG 0.77 5 3.83 8.73  47 A0A078IDV8 BnaC03g69180D protein IIKVITRALRGARRLLKY 0.64 6 3.81 10.16  48 G7MZL5 Interleukin-7 (Fragment) FLKRLLQKIKTCWNKIL 0.76 5 3.79 9.21  49 G7PC28 Interleukin-7 (Fragment) FLKRLLQKIKTCWNKIL 0.76 5 3.79 9.07  50 Q95J83 Interleukin 7 FLKRLLQKIKTCWNKIL 0.76 5 3.79 9  51 Q8HZN1 Interleukin-7 FLKRLLQKIKTCWNKIL 0.76 5 3.79 9  52 B6E124 Interleukin-7 FLKRLLQKIKTCWNKIL 0.76 5 3.79 8.99  53 A0A096N2Z5 Uncharacterized protein FLKRLLQKIKTCWNKIL 0.76 5 3.79 9  54 F7G6P7 Uncharacterized protein (Fragment) FLKRLLQKIKTCWNKIL 0.76 5 3.79 8.81  55 F7G6Q1 Uncharacterized protein (Fragment) FLKRLLQKIKTCWNKIL 0.76 5 3.79 8.71  56 D0NCA0 Putative uncharacterized protein NGLFQKIKRWWKRIFDR 0.75 5 3.73 10.62  57 A0A093GP59 Interferon alpha-2 (Fragment) RRCLQLAHKVIRKL 0.67 5.5 3.69 9.81  58 H0Z3Q1 Uncharacterized protein FIKKLMTFIRKVLKT 0.74 5 3.68 9.41  59 L7NUA5 Interleukin-7 variant 2 FVKRLLDEIKTCWNKILR 0.70 5 3.51 9.56  60 GAKK L7NU76 Interleukin-7 variant 5 FVKRLLDEIKTCWNKILR 0.70 5 3.51 9.48  61 GAKK L7NU80 Interleukin-7 variant 1 FVKRLLDEIKTCWNKILR 0.70 5 3.51 9.33  62 GAKK L7NUC8 Interleukin-7 variant 4 FVKRLLDEIKTCWNKILR 0.70 5 3.51 9.24  63 GAKK L7NU82 Interleukin-7 variant 3 FVKRLLDEIKTCWNKILR 0.70 5 3.51 9  64 GAKK L7NU81 Interleukin-7 variant 6 FVKRLLDEIKTCWNKILR 0.70 5 3.51 8.85  65 GAKK H0VFS1 Uncharacterized protein (Fragment) FLKTLLQKIKTCWNKILRG 0.68 5 3.40 9.27  66 P48816 Lysozyme (EC 3.2.1.17) (1,4- ITKAAKCAKKIYKR 0.56 6 3.37 9.06  67 beta-N-acetylmuramidase) W2I7H3 Uncharacterized protein TQAWTNFKKWFKNWFKK 0.67 5 3.36 8.34  68 W2JZX5 Uncharacterized protein KEVIKKFTKAMEKMKK 0.67 5 3.35 10.12  69 W2VUY9 Uncharacterized protein KEVIKKFTKAMEKMKK 0.67 5 3.35 10.12  70 W2HTE6 Uncharacterized protein KEVIKKFTKAMEKMKK 0.67 5 3.35 10.12  71 A0A080Z0D6 Uncharacterized protein KEVIKKFTKAMEKMKK 0.67 5 3.35 10.12  72 W2FPC4 Uncharacterized protein KEVIKKFTKAMEKMKK 0.67 5 3.35 9.91  73 W2Y3P7 Uncharacterized protein KEVIKKFTKAMEKMKK 0.67 5 3.35 9.91  74 W2QT56 Uncharacterized protein KEVIKKFTKAMEKMKK 0.67 5 3.35 9.91  75 W2N8A5 Uncharacterized protein NGLFQKIKRWWKRIFEH 0.74 4.5 3.34 10.43  76 W2RAE5 Uncharacterized protein NGLFQKIKRWWKRIFEH 0.74 4.5 3.34 10.43  77 W2WX61 Uncharacterized protein NGLFQKIKRWWKRIFEH 0.74 4.5 3.34 10.43  78 A0A081A496 Uncharacterized protein NGLFQKIKRWWKRIFEH 0.74 4.5 3.34 10.31  79 W2Z9T6 Uncharacterized protein NGLFQKIKRWWKRIFEH 0.74 4.5 3.34 10.31  80 P50718 Lysozyme (EC 3.2.1.17) ITKASKCAKKIYKR 0.55 6 3.33 9.15  81 (1,4-beta-N-acetylmuramidase) T0L7V9 Uncharacterized protein LWDKFQKFLKKVIRI 0.82 4 3.30 6.88  82 A0A072TQ32 Transmembrane protein, putative CNHMPNLLTRCLQRLKRLK 0.60 5.5 3.28 10.05  83 G7KJJ5 Nodule Cysteine-Rich (NCR) HSFYKCIDNLCKRFRR 0.73 4.5 3.28 8.83  84 secreted peptide   A0A091WML3 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.78  85 A0A091K0Z9 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.78  86 A0A091JCY0 Interferon epsilon (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.76  87 A0A091KMX5 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.72  88 A0A093RGB3 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.63  89 A0A091SY09 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.62  90 A0A094KQN9 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.62  91 A0A093L2W4 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.6  92 A0A093FC11 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.55  93 A0A087QSI7 Interferon alpha-2 (Fragment) MRRCLQLIDKVIRKL 0.81 4 3.26 9.46  94 A0A087VKA8 Interferon omega-1 (Fragment) MRRCLQFIDKVIRKL 0.81 4 3.26 9.57  95 A0A091QVA3 Interferon omega-1 (Fragment) MRKCLQLIDKVIRKL 0.81 4 3.26 9.65  96 A0A091GAP8 Interferon alpha-2 (Fragment) MRRCLQLIEKVIRKL 0.81 4 3.25 9.87  97 A0A091L4R0 Interleukin-21 (Fragment) KEFLKSFEKLIKKVIR 0.81 4 3.23 9.32  98 A0A059BP64 Uncharacterized protein NLCKRACRTCCTHCRRVP 0.59 5.5 3.22 8.61  99 A0A091VJQ6 Interferon alpha-2 (Fragment) RRCLQLIDKVIRKL 0.80 4 3.19 9.7 100 A5JYR0 Protein C06C3.10 YARRFSTLFRHLIKMI 0.71 4.5 3.18 11.56 101 A0A091JU23 Interleukin-21 (Fragment) REFLKSFAKLIKKVI 0.79 4 3.16 9.41 102 A0A093IE23 Interferon alpha-2 (Fragment) MRRCLQLIDKVMRKL 0.79 4 3.16 9.65 103 A0A091P9G9 Interferon alpha-2 (Fragment) MRRCLQLIDKVVRKL 0.79 4 3.16 9.76 104 A0A091U5M0 Interferon alpha-2 (Fragment) MRRCLQLIDKAIRKI 0.79 4 3.14 9.65 105 A0A093BUH7 Interferon epsilon (Fragment) MRRCLQLIDKAIRKL 0.78 4 3.11 9.33 106 A0A094KTL8 Interferon alpha-2 (Fragment) MRRCLQLIDKAIKKL 0.78 4 3.11 9.55 107 A0A091SBM1 Interferon beta (Fragment) MRRCLQLVDKVIRKL 0.78 4 3.11 9.61 108 A0A093HSG6 Interferon alpha-2 (Fragment) MRRCLQFVDKVIKRL 0.78 4 3.10 9.49 109 A0A091HDU2 Interferon alpha-2 (Fragment) MRKCLQLVDKVIRKL 0.78 4 3.10 9.72 110 G1PT51 Uncharacterized protein VKKAVKYLRTIMKS 0.62 5 3.10 9.96 111 A0A091UC07 Interleukin-21 (Fragment) KEFLKSFAKLIKQVIR 0.78 4 3.10 9.61 112 A0A094K224 Interleukin-21 (Fragment) KEFLKSFAKLIKQVIR 0.78 4 3.10 9.54 113 A0A093IDB2 Interleukin-21 (Fragment) KEFLKSFAKLIKQVIR 0.78 4 3.10 9.52 114 A0A091VRY9 Interleukin-21 (Fragment) KEFLKSFAKLIKQVIR 0.78 4 3.10 9.46 115 O95399 Urotensin-2 (Urotensin II) LSHLLARIWKPYKK 0.68 4.5 3.05 4.37 116 (U-II) (UII) M7W4U4 Lecithin:cholesterol acyltrans- LSIFAKCFHDLIKKFKKLG 0.68 4.5 3.04 4.45 117 ferasefamily protein (Fragment) A0A091M384 Interferon omega-1 (Fragment) MRRCLQLIDKVARKL 0.75 4 3.01 9.62 118 Q8HYR8 IL-7 LLQKIKTCWNKILRGIKE 0.75 4 3.01 9.04 119 L8IFQ4 Interleukin-7 (Fragment) LLQKIKTCWNKILRGIKE 0.75 4 3.01 9.02 120 P26895 Interleukin-7 (IL-7) LLQKIKTCWNKILRGIKE 0.75 4 3.01 9.02 121 P01588 Erythropoietin (Epoetin) FRKLFRVYSNFLRG 0.75 4 3.00 8.75 122 Q91Y32 Interleukin-7 FLKRLLREIKTCWNKILNS 0.74 4 2.98 8.75 123 R0LJN1 Interferon alpha-2 (Fragment) IRRCLQLIDKAVRKLY 0.74 4 2.97 9.5 124 A0A044QPZ9 Uncharacterized protein NFLSLIKKIFSVWKR 0.74 4 2.96 8.47 125 F7BPI0 Uncharacterized protein KIMRKIMTNLSRLC 0.74 4 2.95 10.06 126 A0A093EX75 Interleukin-21 KEFLKSFAKLIQKVIKS 0.72 4 2.90 9.28 127 A0A091E7E9 Interleukin-5 (Fragment) FIKKLMTFIQKVLKN 0.72 4 2.88 9.13 128 P0DKN3 Turripeptide Lol11.2 (OL67) RDAGRLLRSLKKLK 0.57 5 2.87 8.7 129 A0A091LVZ3 Uncharacterized protein KNATTFIKKLMTFIRKA 0.57 5 2.84 9.2 130 S9YX13 Olfactory receptor 4A15 GEMKNAMKKLWTRVRK 0.57 5 2.83 9.46 131 V9DV74 Uncharacterized protein TQAWTNFKKWFKKWFKE 0.69 4 2.74 8.34 132 W2KE58 Uncharacterized protein TQAWTNFKKWFKKWFKE 0.69 4 2.74 8.34 133 L8YAP6 Interleukin-13 EVAQFVKDLLRHLKRLYRH 0.68 4 2.73 8.97 134 Q6UXQ8 Putative uncharacterized protein GAGRKVCAKLVKRL 0.55 5 2.73 11.8 135 UNQ6190/PRO20217 U6CRU1 Interleukin-7 LLQKIKTCWNKILRGS 0.68 4 2.71 9.03 136 D2HR10 Putative uncharacterized protein LLQKIKTCWNKILRGS 0.68 4 2.71 9.7 137 (Fragment) M3W2P1 Uncharacterized protein LLQKIKTCWNKILRGS 0.68 4 2.71 9.15 138 M4CA57 Uncharacterized protein NFIKRINDFLKKAKS 0.67 4 2.69 9.89 139 P13725 Oncostatin-M (OSM) RFLHGYHRFMHSVGRVFS 0.60 4.5 2.69 10.6 140 KWGES U5TZM0 Interleukin-7 LLQKIKTCWNKILRGAKEY 0.67 4 2.68 8.85 141 Q9N2G6 Interleukin-7 (IL-7) LLQKIKTCWNKILRGAKEY 0.67 4 2.68 8.7 142 K7GL89 Uncharacterized protein LLQKIKTCWNKILRGAKEY 0.67 4 2.68 7.77 143 K7GL01 Uncharacterized protein (Fragment) LLQKIKTCWNKILRGAKEY 0.67 4 2.68 7.74 144 A0A093PT39 Interleukin-5 (Fragment) FINKLMTFIRKALKP 0.66 4 2.65 9.3 145 Q91ZL2 Interleukin 4 LKDFLKSLKRIM 0.88 3 2.64 9.27 146 P81278 Prolactin-releasing peptide GRGIRPVGRFGRR 0.53 5 2.63 11.42 147 (PrRP) (Prolactin-releasing hormone) [Cleaved into: Prolactin-releasing peptide PrRP31; Prolactin-releasing peptide PrRP20] A0A080YYV3 Uncharacterized protein NGLWQRFLRWWNRLFH 0.75 3.5 2.62 9.86 148 W2VQZ9 Uncharacterized protein NGLWQRFLRWWNRLFH 0.75 3.5 2.62 9.86 149 W2PXT4 Uncharacterized protein NGLWQRFLRWWNRLFH 0.75 3.5 2.62 9.69 150 P86442 Neuropeptide F (Lom-NPF) (NPF) SADKFWRRFARR 0.66 4 2.62 10.84 151 (longNPF) B8BP19 Putative uncharacterized protein WSRWWSSCWRLWTRIY 0.87 3 2.61 4.85 152 W2ZYU4 Uncharacterized protein YLKEALSRFKSWLKR 0.65 4 2.60 10.07 153 Q9XTN0 Peptidoglycan recognition protein SPGRKLYNQIRRWP 0.65 4 2.59 6.71 154 X6P0L4 Uncharacterized protein KYGKSCEKAVHDLWHIIKKF 0.64 4 2.58 8.2 155 P24355 50S ribosomal protein L36, plastid KVYSSVRKICKSCG 0.64 4 2.57 11.06 156 W2YJS0 Uncharacterized protein TQAWTNFKKWFKKMV 0.64 4 2.55 5.08 157 Q9BBQ2 50S ribosomal protein L36, KIGASVRKICEKCR 0.64 4 2.55 11.79 158 chloroplastic W2HIB3 Uncharacterized protein GGGRDVLTRFKSWFKRVFGK 0.50 5 2.52 8.39 159 P50717 Lysozyme (EC 3.2.1.17) (1,4-beta-N- ITKASTCAKKIFKR 0.50 5 2.52 8.92 160 acetylmuramidase) A0A094LD69 Uncharacterized protein (Fragment) FIEKLMTFIRKALKNAR 0.63 4 2.51 9.3 161 Q6UXT8 Protein FAM150A AYYKRCARLLTRLA 0.63 4 2.51 10.39 162 Q4RU86 Protein FAM150-like AYYKRCARLLTRLA 0.63 4 2.51 9.98 163 I7GA36 Macaca fascicularis brain cDNA SQLRNLIRSVRTVMR 0.62 4 2.49 8.59 164 clone: QtrA-18967, similar to human HSPC039 protein (HSPC039), mRNA, RefSeq: NM 016097.2 A0A093IPB5 Interleukin-5 (Fragment) KNATKFIEKLMTFIRRA 0.62 4 2.48 9.2 165 P81277 Prolactin-releasing peptide YASRGIRPVGRFGRR 0.49 5 2.45 11.6 166 (PrRP) (Prolactin-releasing hormone) [Cleaved into: Prolactin-releasing peptide PrRP31; Prolactin-releasing   peptide PrRP20] P01574 Interferon beta (IFN-beta) HLKRYYGRILHYLKA 0.49 5 2.45 8.9 167 (Fibroblast interferon) Q80UG6 Protein FAM150B TIPAYYKRCARLLTRLA 0.61 4 2.44 9.57 168 B2RZ42 Protein FAM150B TIPAYYKRCARLLTRLA 0.61 4 2.44 9.57 169 Q6UX46 Protein FAM150B TIPAYYKRCARLLTRLA 0.61 4 2.44 9.37 170 P55897 Histone H2A [Cleaved into: VGRVHRLLRKGN 0.54 4.5 2.43 12.41 171 Buforin-1 (Buforin I); Buforin-2 (Buforin II)] (Fragment) P07815 50S ribosomal protein L36, chloro- KVAASVRKICEKCR 0.61 4 2.42 11.64 172 plastic Q95PE0 Putative uncharacterized protein IRKIAQLLNKVADAAKKG 0.60 4 2.41 4.94 173 (Fragment) Q2V364 Putative defensin-like protein 23 GGFCKRFAGGAKKCH 0.54 4.5 2.41 9.14 174 Q3E7A6 Uncharacterized protein YML007C-A, MRFMRRLVRNLQY 0.60 4 2.40 12.18 175 mitochondrial P04567 Mast cell degranulating peptide LPGFIGKICRKI 0.80 3 2.39 9.5 176 (MCD peptide)(MCDP) Q3E821 Uncharacterized protein YBL008W-A ILANIHKIIKAYQR 0.68 3.5 2.38 11.19 177 K4A4B8 Uncharacterized protein IFHGCKKVARVL 0.68 3.5 2.37 9.19 178 P01210 Proenkephalin-A [Cleaved into: LAKRYGGFMKRYGGFMKK 0.58 4 2.34 5.3 179 Synenkephalin; Met-enkephalin MDELY (Opioid growth factor) (OGF); PENK(114-133); PENK(143-183); Met-enkephalin-Arg-Gly-Leu; Leu-enkephalin; PENK(237-258); Met-enkephalin-Arg-Phe] A0A085MM52 Uncharacterized protein QAIRCMQSLFRTVKR 0.58 4 2.33 9.45 180 Q8N729 Neuropeptide W (Preproprotein L8) WRRALRAAAGPLAR 0.58 4 2.33 11.4 181 (hPPL8) [Cleaved into: Neuropeptide  W-23 (NPW23) (hL8); Neuropeptide  W-30 (NPW30) (hL8C)] R0MAQ4 NUDIX hydrolase IKRIIRDVKEIVRA 0.77 3 2.32 6.92 182 A0A093C1K6 Interleukin-21 (Fragment) KEFLKSFEKLIQKVIR 0.77 3 2.32 9.21 183 R0M641 Uncharacterized protein EKVSNWWKRIWEKIKN 0.77 3 2.31 5.8 184 R0M5R6 Uncharacterized protein EKVSNWWKRIWEKIKN 0.77 3 2.31 5.8 185 M4BXP7 Uncharacterized protein IPSCVTRALSRAARSIWDA 0.58 4 2.31 9.89 186 LKKI Q9NRR1 Cytokine-like protein 1 (Protein C17) ARKLYTIMNSFCRR 0.58 4 2.31 8.22 187 P81447 Glycosylation-dependent cell GKLMELGHKIMKNLENTVKEI 0.66 3.5 2.29 5.13 188 adhesion molecule 1 (GlyCAM-1) IKYLKSLF (28 kDa milk glycoprotein PP3) (Lactophorin) (Proteose-peptone component 3) (PP3) K7GRI1 Interleukin KPIKEFLERLKSLIQKMIHQ 0.65 3.5 2.27 9.39 189 F1RQN2 Interleukin KPIKEFLERLKSLIQKMIHQ 0.65 3.5 2.27 9.39 190 Q76LU6 Interleukin-21 (IL-21) KPIKEFLERLKSLIQKMIHQ 0.65 3.5 2.27 9.25 191 A0A093G4B0 Interleukin-21 (Fragment) KEFLKSFAKLIQKVI 0.76 3 2.27 9.49 192 A0A091LQM6 Interleukin-21 (Fragment) KEFLKSFAKLIQKVI 0.76 3 2.27 9.22 193 Q09GK2 C-type natriuretic peptide (CNP) RRLKGVAKKGLG 0.45 5 2.27 8.9 194 H2QWB6 Interleukin 7 (Uncharacterized protein) FLKRLLQEIKTCWNKIL 0.75 3 2.26 8.72 195 P13232 Interleukin-7 (IL-7) FLKRLLQEIKTCWNKIL 0.75 3 2.26 8.72 196 Q5FBX5 IL7 nirs variant 1 (Interleukin-7) FLKRLLQEIKTCWNKIL 0.75 3 2.26 6.14 197 A8K673 cDNA FLJ75417, highly similar to FLKRLLQEIKTCWNKIL 0.75 3 2.26 8.72 198 Homo sapiens interleukin 7, mRNA H2PQM2 Uncharacterized protein FLKRLLQEIKTCWNKIL 0.75 3 2.26 8.72 199 G1QNL2 Uncharacterized protein FLKRLLQEIKTCWNKIL 0.75 3 2.26 8.72 200 G3QDY6 Uncharacterized protein FLKRLLQEIKTCWNKIL 0.75 3 2.26 7.75 201 F6TDR4 Interleukin KPLKEFLERLKSLIQKMIHQ 0.64 3.5 2.26 9.66 202 A0A093EHY0 Interleukin-21 (Fragment) KEFLKSFANLIKQVIR 0.75 3 2.26 9.1 203 L8I6N0 Glycosylation-dependent cell GKLMELGHKIMRNLENTVKE 0.56 4 2.25 5.98 204 adhesion molecule 1 TIKYLKSLFSHASEVVK G3KFL3 PexRD2 HKMKKLLRYLNY 0.50 4.5 2.24 9.52 205 Q0ZCA0 Putative accessory gland protein  KSFSSICKELCKLCQRC 0.75 3 2.24 8.7 206 (Fragment) Q0ZC97 Putative accessory gland protein  KSFSSICKELCKLCQRC 0.75 3 2.24 8.64 207 (Fragment) A0A091H9P3 Interleukin-21 (Fragment) KEFLQSFGKLINKVIR 0.74 3 2.23 9.49 208 A0A091V0J9 Myelomonocytic growth factor (Fragment) ILANFQRFLETAYRALRHLAR 0.64 3.5 2.22 5.67 209 A0A091J8M6 Interleukin-21 (Fragment) KEFLKSFAKLIQQVIR 0.74 3 2.22 9.54 210 A0A091K4W3 Interleukin-21 (Fragment) KEFLKSFAKLIQQVIR 0.74 3 2.22 9.39 211 A0A093PBX6 Interleukin-21 (Fragment) KEFLKSFAKLIQQVIR 0.74 3 2.22 9.34 212 A0A091SVN6 Interleukin-21 (Fragment) KEFLKSFAKLIQQVIR 0.74 3 2.22 9.32 213 U3IH11 Interleukin KEFLQSFSKLMKKV 0.74 3 2.22 9.37 214 A0A078J1N0 BnaC02g44190D protein KPLHTMVNHISRFATKMT 0.55 4 2.21 4.97 215 B3NKZ6 Protein Turandot E (Protein Victoria) GGKLFDVLKKIIKVI 0.74 3 2.21 9.62 216 A0A087VKZ4 Interleukin-21 (Fragment) KEFLQSFAKLIKQVIR 0.73 3 2.19 9.34 217 Q28540 Interleukin-7 (IL-7) LLQKIKTCWNKILRGITE 0.73 3 2.19 8.89 218 B9HA59 Uncharacterized protein RIRDVFSSIFRNIFNLFR 0.73 3 2.19 10.7 219 B6E474 8 kDa glycoprotein QKIAQLVKKWIKTVLEAR 0.54 4 2.17 9.78 220 P80195 Glycosylation-dependent cell GKLMELGHKIMRNLENTVKE 0.54 4 2.15 5.98 221 adhesion molecule 1 (GlyCAM-1) TIKYLKSLFSHAFEVVKT (28 kDa milk glycoprotein PP3) (Lactophorin) (Proteose-peptone component 3) (PP3) S7MSM8 Interleukin-13 EVTRLAKDLLQQLRKGVRQGK 0.54 4 2.15 9.4 222 L5M5Z7 Interleukin-13 EVTRLAKDLLQQLRKGVRQGK 0.54 4 2.15 9.1 223 P0CY42 ATP synthase protein 8 (A6L) LPRLLRTYISRI 0.71 3 2.14 10.28 224 (F-ATPase subunit 8) P0CY41 ATP synthase protein 8 (A6L) LPRLLRTYISRI 0.71 3 2.14 10.28 225 (F-ATPase subunit 8) K0KI76 Putative secreted protein LKKLNQIVQKFHQLLQG 0.61 3.5 2.14 4.85 226 T1PF69 Dynein heavy chain and region D6 DFKNLIRKCVDVARKV 0.71 3 2.14 8.71 227 of dynein motor W2HGG0 Uncharacterized protein NATTYLKEALSRFKSWLKR 0.53 4 2.13 10.07 228 V9FSC7 Uncharacterized protein NATTYLKEALSRFKSWLKR 0.53 4 2.13 10.07 229 A0A081AW62 Uncharacterized protein NATTYLKEALSRFKSWLKR 0.53 4 2.13 9.98 230 P36925 Interleukin-8 (IL-8) (C-X-C KWVQKVVQAFLKRAEK 0.53 4 2.12 9.03 231 motif chemokine 8)(Chemokine (C-X-C motif) ligand 8) K7FKJ3 Uncharacterized protein ARKMVTRALRALQN 0.53 4 2.12 8.24 232 F7I3A3 Interleukin KEFLERFKSLLQKMIHR 0.60 3.5 2.11 9.25 233 Q9TJR5 50S ribosomal protein L32, plastid AKKALAKAKTVLK 0.42 5 2.10 11.45 234 W2IIM7 Uncharacterized protein NGLWQRFLRWWNRLFTG 0.70 3 2.10 10.1 235 W2VQV4 Uncharacterized protein NGLWQRFLRWWNRLFTG 0.70 3 2.10 10.1 236 V9EMI9 Uncharacterized protein NGLWQRFLRWWNRLFTG 0.70 3 2.10 10.1 237 D0N532 Putative uncharacterized protein ATKLSDAMQRIKTMFRNWYQK 0.52 4 2.09 10.19 238 P46690 Gibberellin-regulated protein 4 CITFCNKCCRKCL 0.70 3 2.09 9.42 239 (GAST1 protein homolog 4) C5YGU9 Putative uncharacterized protein  CCDLIRKCWHDIKECWKRC 0.83 2.5 2.08 9.02 240 Sb07g003410 Q9GKA2 Erythropoietin KLFRIYSNFLRG 0.69 3 2.08 8.89 241 Q6H8T1 Erythropoietin KLFRIYSNFLRG 0.69 3 2.08 8.74 242 Q6H8S9 Erythropoietin KLFRIYSNFLRG 0.69 3 2.08 8.74 243 Q6H8T0 Erythropoietin KLFRIYSNFLRG 0.69 3 2.08 8.74 244 P33708 Erythropoietin KLFRIYSNFLRG 0.69 3 2.08 8.68 245 Q6H8T2 Erythropoietin KLFRIYSNFLRG 0.69 3 2.08 8.42 246 Q867B1 Erythropoietin KLFRIYSNFLRG 0.69 3 2.08 8.35 247 D7KU31 Putative uncharacterized protein EKFESCMKKCSKICNK 0.69 3 2.08 6.62 248 Q0Z956 Erythropoietin KLFRVYSNFLRG 0.69 3 2.08 9.03 249 P29676 Erythropoietin KLFRVYSNFLRG 0.69 3 2.08 8.73 250 Q28513 Erythropoietin KLFRVYSNFLRG 0.69 3 2.08 8.35 251 P07865 Erythropoietin KLFRVYSNFLRG 0.69 3 2.08 8.35 252 P38215 Putative uncharacterized protein  RKLYHRVGTCIQNIF 0.59 3.5 2.07 6.07 253 YBR013C P06759 Apolipoprotein C-III (Apo-CIII) FKSLKGYWSKFT 0.69 3 2.07 4.36 254 (ApoC-III) (Apolipoprotein C3) B1GVZ6 Interleukin 7 ISSLRSCWNKFEKIISR 0.69 3 2.06 8.91 255 A8MRM1 Protein RALF-like 16 VNHYHRGCEKITRCARDA 0.51 4 2.06 8.45 256 ARY U3K1R5 Uncharacterized protein ISSLRSCWNKFEKLISR 0.69 3 2.06 8.91 257 R4WCP1 Cysteine rich secreted protein CCRKVVKYWTHCCQEGLRCT 0.59 3.5 2.06 8.72 258 C1MNQ9 Predicted protein GRTFRSCARTAFRTAKL 0.41 5 2.05 6.79 259 P07321 Erythropoietin KLFRVYANFLRG 0.68 3 2.05 7.87 260 A0A061DMT2 Uncharacterized protein YGRGCRRCCSYL 0.68 3 2.05 7.78 261 F6PXB2 Uncharacterized protein FLKRLLQEIKTCWNKILSG 0.68 3 2.03 8.71 262 F6PXS2 Uncharacterized protein (Fragment) FLKRLLQEIKTCWNKILSG 0.68 3 2.03 6.91 263 F6PXG8 Uncharacterized protein (Fragment) FLKRLLQEIKTCWNKILSG 0.68 3 2.03 5.68 264 A0A094ICS8 Uncharacterized protein SVARHAARDLRRWM 0.58 3.5 2.01 9.67 265 F6U832 Uncharacterized protein LLQKIKTCWNKILRDAKE 0.67 3 2.01 8.7 266 P09641 Peptide YY-like (PYY) WAKYHAAVRHYVNLITR 0.50 4 2.01 8.39 267 P81028 Peptide YY-like (PYY) WAKYHAAVRHYVNLITR 0.50 4 2.01 8.35 268 G3VIZ8 Uncharacterized protein EEVFKKFKKVAKSFG 0.67 3 2.01 7.83 269 O61466 FMRFamide-like neuropeptides 5 FGRAGAKFIRFGRS 0.50 4 2.00 11.17 270 [Cleaved into: APKPKFIRF-amide; AGAKFIRF-amide; GAKFIRF-amide] C4V9A1 Putative uncharacterized protein DLKTLFTTIWKFIKK 0.67 3 2.00 6.31 271 P86730 Perlwapin (Fragment) KKCCGGCPRLCEK 0.67 3 2.00 8.45 272 A8MQM7 Protein RALF-like 15 KQVANPYRRGCGTIERCR 0.49 4 1.97 9.96 273 H3FNG9 Uncharacterized protein SDAKKLMRDLKRML 0.66 3 1.97 5.38 274 Q29614 Beta-lactoglobulin (Beta-LG) NKGMNEFKKILRTLA 0.65 3 1.95 5.56 275 Q7M747 Secretoglobin family 2B member FTKIKDALKKISQ 0.65 3 1.95 6.11 276 24 (Allergen dI chain C2B) (Androgen-binding protein zeta) (Lacrimal androgen-binding protein zeta) P15457 2S seed storage protein 1 (2S VRKIYQTAKHLP 0.56 3.5 1.95 10.44 277 albumin storage protein) (NWMU2-2S albumin 1) [Cleaved into: 2S seed storage protein 1 small subunit; 2S seed storage protein 1 large subunit] Q0V822 Protein RALF-like 26 VHNYSRGCSRITRCR 0.43 4.5 1.95 11.19 278 J9P346 Uncharacterized protein KIKRIVQKILESAER 0.65 3 1.95 8.73 279 P49164 50S ribosomal protein L34, chloroplastic ARMATKLGRKVLN 0.48 4 1.93 12.23 280 A5WYF5 Putative 4.8 kDa secreted salivary  KTLVNLWSKLAQRIF 0.64 3 1.93 4.53 281 gland protein Q9GZZ8 Extracellular glycoprotein lacritin QALAKAGKGMHGGVPGGKQ 0.43 4.5 1.93 5.17 282 FIENGSEFAQKLLKKF Q9FZA0 Protein RALF-like 4 NPYRRGCSAITHCYRYAR 0.43 4.5 1.92 10.76 283 K9JHY4 Leptin RVKQVLNQLLKNMDHLK 0.55 3.5 1.92 9.15 284 A0A076YJS8 Interleukin 4/13-3 VSSFLEHVKRCVRH 0.63 3 1.90 8.59 285 A0A023G9C2 Putative secreted protein LRTVTELLKKVVDAAKK 0.63 3 1.90 5.02 286 B4G535 Protein Turandot Z AKALFNKLTEYLKK 0.63 3 1.90 5.25 287 B1MTQ2 Interleukin 13 (Predicted) EVAQFVKDLLRHLRKLFHQG 0.63 3 1.89 9.02 288 B0KWP3 Interleukin 13 (Interleukin 13  EVAQFVKDLLRHLRKLFHQG 0.63 3 1.89 8.78 289 (Predicted)) F7H944 Uncharacterized protein (Fragment) EVAQFVKDLLRHLRKLFHQG 0.63 3 1.89 8.69 290 E9Q6L3 Methylglutaconyl-CoA hydratase,  KNLLKMLSKAVDALKS 0.62 3 1.87 9.72 291 mitochondrial Q9P0W0 Interferon kappa (IFN-kappa) LRRYFHRINFLKE 0.74 2.5 1.86 8.8 292 O82377 EMBRYO SURROUNDING FACTOR 1-like SRCYRSLYKCVA 0.62 3 1.85 8.9 293 protein 6 G3V2A7 Type II iodothyronine deiodinase QWLTSCWSTLMRLIHQMA 0.53 3.5 1.84 10.86 294 GRYR P48131 50S ribosomal protein L36, cyanelle ASVRKMCEKCRTIR 0.46 4 1.84 11.64 295 Q43194 Non-specific lipid-transfer protein CLKNAARGIRGLN 0.61 3 1.83 9.45 296 2 (LTP 2) Q0IQK9 Non-specific lipid-transfer protein CLKNAARGIKGLN 0.61 3 1.83 9.41 297 1 (LTP 1) (PAPI) B5FW67 Interleukin 13 (Predicted) EVAQFIKDLLRHLKK 0.72 2.5 1.81 9.42 298 (Uncharacterized protein) P58784 Trp-Contryphan-P GGAIGKFMNVLRR 0.60 3 1.79 3.8 299 O65919 Protein RALF-like 10 ANEYRRGCSKITRCK 0.45 4 1.79 9.69 300 P0C0P7 Neuropeptide S RSFRNGVGSGVKK 0.45 4 1.78 12.31 301 G2TRK4 Uncharacterized protein C1399.06 TILRKARNLLNHGI 0.51 3.5 1.78 10.3 302 P86941 Insulinotropic peptide 1 (FSIP) AVWKDFLKNIGKAAGKAV 0.59 3 1.78 8.47 303 B4MKX9 GK17255 KHAHSMITSMLSFVNSVVN 0.44 4 1.78 8.14 304 FGRSFVRN A0A016W2T3 Uncharacterized protein VKKMLKDVPKLATS 0.59 3 1.78 8.28 305 Q5UW37 VEGF coregulated chemokine 1(13.6  RACQQFLKRCHL 0.51 3.5 1.77 10.37 306 kDa protein) (C-X-C motif chemokine 17) Q9GLK4 Natriuretic peptides B (Gamma- NVLRALRRLGSS 0.59 3 1.77 7.09 307 brain natriuretic peptide) [Cleaved into: Brain natriuretic peptide 32(BNP-32)] A0A074ZW10 Uncharacterized protein GKNWSEFVRSMLRAMSKVAA 0.59 3 1.77 5.07 308 G3T2N9 Uncharacterized protein ELITFMKNLLDHLRRIYR 0.71 2.5 1.76 9.23 309 Q2XPU9 Vdg3 ICKSFQSLVHRFGHVT 0.58 3 1.75 5.32 310 P22298 Antileukoproteinase (ALP) (Secretory LKCCKSMCGKVC 0.58 3 1.75 9.11 311 leukocyte protease inhibitor) (Fragment) O77234 SPO-1 protein (Stage-specific  KRMEYIAKKLDK 0.58 3 1.74 8.92 312 protein SPO-1) (Stathmin-like protein) H2MKX6 Uncharacterized protein ASVVQQIQRIIRVLRS 0.58 3 1.73 11.83 313 P15696 Interferon tau-1 (IFN-tau-1) KRLRKMGGDLNSL 0.58 3 1.73 5.74 314 (Antiluteolysin) (Trophoblast anti-   luteolytic protein) (Trophoblast  protein 1) (TP-1) (Trophoblastin) M4WK63 Cul o 5 allergen KSYVEKLTKALSTIRQCI 0.57 3 1.72 9.23 315 A8CL69 PBAN-type neuropeptides RLGRQLHNIVDK 0.69 2.5 1.72 5.5 316 (Pheromone/pyrokinin biosynthesis- activating neuropeptide) [Cleaved into: TSQDITSGMWFGPRL-amide (Pyrokinin-1); QITQFTPRL-amide (Pyrokinin-2); IYLPLFASRL-amide; VPWTPSPRL-amide] F6R9B2 Uncharacterized protein (Fragment) SQLTNLIRSVRTVMR 0.57 3 1.71 8.95 317 Q3SYR5 Apolipoprotein C-IV (Apo-CIV)(ApoC-IV) LKDLGSRARAWLRS 0.57 3 1.71 9.07 318 (Apolipoprotein C4) P0C0P5 Neuropeptide S RSFRNGVGTGMKK 0.43 4 1.71 12.31 319 P0C0P6 Neuropeptide S RSFRNGVGTGMKK 0.43 4 1.71 12.02 320 Q6NME6 Protein RALF-like 19 YRRGCSVITHCYRQ 0.49 3.5 1.71 10.75 321 P07597 Non-specific lipid-transfer protein CLKGIARGIHNLN 0.68 2.5 1.70 8.19 322 1 (LIP 1) (Probable amylase/protease inhibitor) Q9WVA9 Pro-FMRFamide-related neuropeptide FF RFGRNAWGPWSK 0.56 3 1.69 9.75 323 (FMRFamide-related peptides) [Cleaved into: Neuropeptide SF (NPSF); Neuropeptide FF (NPFF); Neuropeptide AF-like (NPAF)] Q9SRY3 Protein RALF-like 1 (Rapid ANPYSRGCSKIARCR 0.42 4 1.69 10.06 324 alkalinization factor 1) (AtRALF1) A0A024GHZ6 Albugo candida WGS project ILHSFLKVLHHL 0.67 2.5 1.69 9.18 325 CAIX00000000 data, strain Ac Nc2, contig AcNc2_CONTIG_128_length_64418 Q91159 Lysozyme C(EC 3.2.1.17) (1,4-beta-N- IKCAKKIARDAHGLT 0.48 3.5 1.69 5.11 326 acetylmuramidase C) Q5DF01 SJCHGC01792 protein VLKLWNSVLKHICNLS 0.67 2.5 1.69 4.41 327 B5L332 CSF3 (Uncharacterized protein) LRQLRSFMQDVFRSLRC 0.56 3 1.68 8.12 328 Q70PW6 Peptidoglycan-recognition protein SB2 GRHLLNELKKWP 0.67 2.5 1.68 11.06 329 (EC 3.5.1.28) P24296 Non-specific lipid-transfer protein NCLKGIARGIHNLN 0.67 2.5 1.67 8.2 330 (LTP) (Phospholipid transfer protein)  (PLTP) (ns-LTP1) (Fragment) K7M000 Uncharacterized protein WRRVMHCFSYCW 0.67 2.5 1.67 8.35 331 P0C0P8 Neuropeptide S RSFRNGVGSGAKK 0.42 4 1.66 12.31 332 B9RZ82 Putative uncharacterized protein DLTRALHDLVKALKKAYR 0.66 2.5 1.66 5.64 333 TLD O46633 Interferon tau (IFN-tau) (Antiluteolysin) KRLRKMGGDLNS 0.55 3 1.66 6.57 334 (Trophoblast antiluteolytic protein)  (Trophoblast protein 1) (TP-1) (Trophoblastin) A0A093R5C1 Uncharacterized protein (Fragment) KNATTFIEKLMTFIRKA 0.55 3 1.66 7.99 335 M3VWX3 Uncharacterized protein (Fragment) EVIQLVKNLLNHLRR 0.66 2.5 1.65 9.42 336 G1T948 Interleukin (Fragment) LKEFLERLKSLIQKMIHQ 0.66 2.5 1.65 9.67 337 O64466 Protein RALF-like 11 VNEYSRGCSKIHRCR 0.47 3.5 1.65 8.5 338 F4ISE1 Protein RALF-like 12 VNEYSRGCSKIHRCR 0.47 3.5 1.65 8.5 339 F4ISE2 Protein RALF-like 13 VNEYSRGCSKIHRCR 0.47 3.5 1.65 8.5 340 M4QCE6 Adipokinetic 3 CGQFTRLCRHFVHELKQ 0.55 3 1.65 6.45 341 ALTS E9NX77 AKH/corazonin-like hormone CGQFTRLCRHFVHELKQ 0.55 3 1.65 6.44 342 ALTS Q9XSN5 Apolipoprotein C-I (Apo-CI) (ApoC-I) DKAKKAIERIKQ 0.55 3 1.64 9.11 343 (Apolipoprotein C1) [Cleaved into: Truncated apolipoprotein C-I] R0MG91 Uncharacterized protein EKISDWWKRIWEKIKN 0.82 2 1.63 5.34 344 P02815 Mucin-like protein 2 (16.5 kDa sub- LENMKTVIKSGVEKLKNF 0.54 3 1.63 3.8 345 mandibular gland glycoprotein) (Salivary LQRG protein 1) A0A087VIJ1 Uncharacterized protein NATRFMEKLMTFVRKA 0.54 3 1.62 8.87 346 Q4G392 50S ribosomal protein L34, chloroplastic RMATKAGRRVINAR 0.32 5 1.62 12.96 347 U3JCF4 Uncharacterized protein (Fragment) ILANFQRFLETAYRALRHL 0.64 2.5 1.61 5.39 348 Q9WVA8 Pro-FMRFamide-related neuropeptide FF RFGRSAWGSWSK 0.54 3 1.61 9.47 349 (FMRFamide-related peptides) [Cleaved  into: Neuropeptide SF (NPSF); Neuro- peptide FF (NPFF); Neuropeptide  AF-like (NPAF)] A0A061HZA0 Ras-related and estrogen-regulated THVKQAINKMLTKISS 0.46 3.5 1.61 5.48 350 growth inhibitor-like protein A8XRC0 Protein CBG17503 RELPKVCRNIFSRVSL 0.53 3 1.60 5.86 351 Q9TLU9 50S ribosomal protein L36, chloroplastic ASVRKICSRCVALK 0.40 4 1.60 11.48 352 P06833 Caltrin (Calcium transport inhibitor) LSRYAKLANRLANP 0.53 3 1.60 10.36 353 (Peptide YY-2) (Peptide YY2) (Seminal- plasmin) (SPLN) V5IDJ5 Putative secreted protein (Fragment) DILRECAKGLEVRVAENQH 0.35 4.5 1.60 5.86 354 LANETVEYFFKKLWRGVKK VVKK Q94CG1 Glycine rich protein YGCCRKGYNGCKRCCSYAG 0.53 3 1.59 8.79 355 EAIDKV D2K2T8 Glycine-rich protein YGCCRKGYNGCKRCCSYAG 0.53 3 1.59 8.79 356 EAIDKV G1NDN8 Interleukin QEFLNSFSKLMQKVIKIH 0.64 2.5 1.59 9.07 357 A4K2W7 WAP four-disulfide core domain protein 5 RCLSPMNHLCHK 0.53 3 1.59 8.73 358 A4K2T2 WAP four-disulfide core domain protein 5 RCLSPMNHLCHK 0.53 3 1.59 8.73 359 F9X658 Uncharacterized protein AFNAVKRLLDQARQLGR 0.53 3 1.58 9.3 360 V5H076 Putative secreted protein AAKNALQKFIEEMKKIK 0.53 3 1.58 6.14 361 W2XXG7 Uncharacterized protein EVKKIWATIIERLYKLWRDW 0.79 2 1.57 9.82 362 P54615 Osteocalcin-related protein (Gamma- GLKTAYRRIYGI 0.52 3 1.57 4.44 363 carboxyglutamic acid-containing pro- tein 3) (Nephrocalcin) (OC-X) P86546 Osteocalcin (Bone Gla protein) (BGP) GLKTAYKRIYGI 0.52 3 1.56 4.44 364 (Gamma-carboxyglutamic acid-containing protein) P86547 Osteocalcin-2 (Bone Gla protein 2) GLKTAYKRIYGI 0.52 3 1.56 4.44 365 (BGP2) (Gamma-carboxyglutamic acid- containing protein 2) V9NK57 IL-4 delta 3 LKDFLENLKRIMQK 0.78 2 1.56 9.81 366 V5H5J7 Putative secreted protein QVLHQVQKLANELLRKL 0.62 2.5 1.56 9.69 367 Q8GWK5 Gibberellin-regulated protein 9  CHRACGSCCAKC 0.62 2.5 1.56 9.36 368 (GAST1 protein homolog 9) P81765 Tyrosinase inhibitor (Phenol oxidase  QCLANGSKCYSHDVCCTKR 0.31 5 1.56 8.32 369 inhibitor) (Phenoloxidase inhibitor) (POI) CHNYAKKCVT A0A091LH39 Uncharacterized protein (Fragment) KSATTFIEKLTTFIRKAS 0.52 3 1.55 8.98 370 Q7M1H2 Glycine-rich protein KGYYKGCKKCCSYAGQAMD 0.52 3 1.55 9.28 371 KVTE A0A091FKF3 Myelomonocytic growth factor (Fragment) ILANFQRFLETAYRALRHLA 0.62 2.5 1.55 6.57 372 G5E3B9 Putative udp-n-acetyl-alpha-d- EGWSTLMRTVHSVIKR 0.62 2.5 1.55 5.04 373 galactosamine:polypeptide n- acetylgalactosaminyltransferase (Fragment) D7MAD4 Putative uncharacterized protein DLSSVAKTLIHRLHK 0.51 3 1.54 4.77 374 P23137 Glycine-rich protein YGCCRKGYNGCKRCCSYAG 0.51 3 1.53 8.29 375 EAMDKV Q9FXS8 Glycine rich protein (Glycine-rich protein) YGCCRKGYNGCKRCCSYAG 0.51 3 1.53 8.27 376 EAMDKV Q9LWA1 Cell wall protein (Glycine-rich protein) YGCCRKGYNGCKRCCSYAG 0.51 3 1.53 7.8 377 EAMDKV P15460 2S seed storage protein 4 (2S albumin  VRKIYQAAKYLP 0.51 3 1.53 10.32 378 storage protein) (NWMU2-2S albumin 4) [Cleaved into: 2S seed storage protein 4 small subunit; 2S seed storage protein 4 large subunit] A9RBU6 Uncharacterized protein ASQLLKVLHKIWTQVP 0.61 2.5 1.53 7.13 379 Q4XR40 Putative uncharacterized protein (Fragment) VALVTGAGRGIGRSIAKTL 0.34 4.5 1.52 9.95 380 AKSVSHVL P01740 T-cell receptor gamma chain V region V108A RWSSGFHKVFAEGTKLI 0.61 2.5 1.52 7.92 381 G1LH25 Interleukin KEFLERLKSLIQRMIHQ 0.60 2.5 1.50 9.66 382 X2FJF4 Interleukin-4 (Fragment) KLSNMLRNLMHLVNQ 0.60 2.5 1.50 6.13 383 I3SFC2 Uncharacterized protein RVIKCIDHICQYARNL 0.60 2.5 1.50 6.92 384 A7KHG0 Nodule Cysteine-Rich (NCR) secreted  RVIKCIDHICQYARNL 0.60 2.5 1.50 6.06 385 peptide (Nodule-specific cysteine-rich peptide 330) H2PE90 Interleukin KEFLERFKSLLQKMIHQ 0.60 2.5 1.50 9.42 386 H2QQ45 Interleukin KEFLERFKSLLQKMIHQ 0.60 2.5 1.50 9.42 387 G1RE48 Interleukin KEFLERFKSLLQKMIHQ 0.60 2.5 1.50 9.42 388 G3QY93 Interleukin KEFLERFKSLLQKMIHQ 0.60 2.5 1.50 9.42 389 Q9HBE4 Interleukin-21 (IL-21) (Zall) KEFLERFKSLLQKMIHQ 0.60 2.5 1.50 9.42 390 F7EGQ8 Interleukin KEFLERFKSLLQKMIHQ 0.60 2.5 1.50 9.12 391 G7P680 Interleukin KEFLERFKSLLQKMIHQ 0.60 2.5 1.50 9.12 392 A0A096N144 Uncharacterized protein KEFLERFKSLLQKMIHQ 0.60 2.5 1.50 9.12 393 M3W5A3 Interleukin KEFLERLKSLIQKMIHQ 0.60 2.5 1.50 9.51 394 Q6L7I9 Interleukin-21 (IL-21) KEFLERLKSLIQKMIHQ 0.60 2.5 1.50 9.42 395 A2D655 IL10 (Fragment) HMLRDLRDAFSRVKTFF 0.60 2.5 1.49 9.49 396 A2D4R8 IL10 (Fragment) HMLRDLRDAFSRVKTFF 0.60 2.5 1.49 9.49 397 A2T6I1 IL10 (Fragment) HMLRDLRDAFSRVKTFF 0.60 2.5 1.49 9.49 398 G3SXC9 Interleukin KEFLERLKSLLQKMIHQ 0.60 2.5 1.49 9.73 399 A0A078GU36 BnaCnng05800D protein KKAASVIQKILKDFGL 0.49 3 1.48 9.75 400 M4FHC5 Uncharacterized protein KKAASVIQKILKDFGL 0.49 3 1.48 9.75 401 Q2V2Y3 Putative defensin-like protein 84 CMKKGGGHCQAYIGR 0.42 3.5 1.48 8.88 402 Q6V9X0 Antileukoproteinase (ALP) (Secretory GALKCCKAMCGKVC 0.49 3 1.47 8.6 403 leukocyte protease inhibitor) E0W4Y8 Avh313 KTLARWAQNAFSKLL 0.49 3 1.47 10.14 404 G5A7V2 Putative uncharacterized protein KTLARWAQNAFSKLL 0.49 3 1.47 10.14 405 I3MIK7 Interleukin KEFLERLKSLLQKMVHQ 0.59 2.5 1.46 9.69 406 P0DKV5 Apolipoprotein C-I (Apo-CI) (ApoC-I) DKVREFFKRIKE 0.73 2 1.46 8.28 407 (Apolipoprotein C1) [Cleaved into:  Truncated apolipoprotein C-I] Q66KU1 C-type lectin domain family 3 member GKWVDEVCRSLKKYI 0.73 2 1.46 8.96 408 A homolog (C-type lectin superfamily  member 1 homolog) A0A078FAD2 BnaA09g39310D protein SAMERLNNWLKTFKH 0.58 2.5 1.45 7.98 409 H0GIJ2 YJL160C-like protein VVSHIVSQIGDGQLQITTA 0.32 4.5 1.44 9.06 410 KKCCHKVHNCCSK L7JS03 Uncharacterized protein (Fragment) RRCVNNFDDVFNSVFRKL 0.72 2 1.44 7.82 411 D7REK7 Interleukin-4 STLRDFLERLKKIMKE 0.72 2 1.44 9.46 412 Q865X5 Interleukin-4 (IL-4) (B-cell stimulatory  STLRDFLERLKKIMKE 0.72 2 1.44 9.46 413 factor 1) (BSF-1) (Lymphocyte  stimulatory factor 1) P51744 Interleukin-4 (IL-4) (B-cell stimulatory  LKNLLERLKTIMKE 0.72 2 1.44 9.51 414 factor 1) (BSF-1) (Lymphocyte stimulatory  factor 1) Q9EQ14 Interleukin-23 subunit alpha (IL-23 SKILRSLQAFLAIAARV 0.41 3.5 1.43 5.85 415 subunit alpha) (IL-23 -A) (Interleukin-   FAHGAA 23 subunit p19) (IL-23p19) Q91Z84 Interleukin-23 subunit alpha (IL-23   SKILRSLQAFLAIAARV 0.41 3.5 1.43 5.63 416 subunit alpha) (IL-23 -A) (Interleukin-23   FAHGAA subunit p19) (IL-23p19) P06307 Cholecystokinin (CCK) [Cleaved into: AHLGALLARYIQQARKA 0.41 3.5 1.43 9.7 417 Cholecystokinin-58 (CCK58); Cholecysto- kinin-58 desnonopeptide ((1-49)-CCK58);  Cholecystokinin-39 (CCK39); Chole- cystokinin-33 (CCK33); Cholecystokinin-  25 (CCK25); Cholecystokinin-18 (CCK18); Cholecystokinin-12 (CCK12); Chole- cystokinin-8 (CCK8); Cholecystokinin-7 (CCK7); Cholecystokinin-5 (CCK5)] Q3E7Z9 Uncharacterized protein YOL038C-A MKYMGSFLRKAAT 0.48 3 1.43 11.76 418 P32648 VIP peptides [Cleaved into: Intestinal  AVKKYLNSILNGK 0.48 3 1.43 6.76 419 peptide PHI-42; Intestinal peptide PHI-27 (Peptide histidine isoleucinamide 27); Vasoactive intestinal peptide (VIP) (Vasoactive intestinal polypeptide)] P01282 VIP peptides [Cleaved into: Intestinal  AVKKYLNSILNGK 0.48 3 1.43 6.76 420 peptide PHV-42 (Peptide histidine valine  42); Intestinal peptide PHM-27 (Peptide  histidine methioninamide 27); Vasoactive  intestinal peptide (VIP) (Vasoactive  intestinal polypeptide)] P01283 VIP peptides [Cleaved into: Intestinal  AVKKYLNSILNGK 0.48 3 1.43 6.76 421 peptide PHV-42; Intestinal peptide PHI-27  (Peptide histidine isoleucinamide 27);   Vasoactive intestinal peptide (VIP)  (Vasoactive intestinal polypeptide)] P81401 VIP peptides [Cleaved into: Intestinal  AVKKYLNSILNGK 0.48 3 1.43 6.75 422 peptide PHI-27 (Peptide histidine iso-  leucinamide 27); Vasoactive intestinal   peptide (VIP) (Vasoactive intestinal poly- peptide)] Q76LU5 Interleukin-21 (IL-21) KEYLERLKSLIQKMIHQ 0.57 2.5 1.43 9.46 423 C7BVU1 Putative DNA-binding response regulator KNVVEDIKKILQKMW 0.71 2 1.43 4.56 424 CreB Q2V4J2 Putative defensin-like protein 26 QRCNRWCHNGCGNGKGGYY 0.29 5 1.43 8.46 425 KSMSHGGQ H0VLF2 Interleukin REFLERMKSLLQKMIHQ 0.57 2.5 1.42 9.61 426 D2HN52 Interleukin (Fragment) KEFLERLKSLIQRV 0.71 2 1.42 9.66 427 P01213 Proenkephalin-B (Beta-neoendorphin- EDLYKRYGGFLRRI 0.71 2 1.42 6.1 428 dynorphin) (Preprodynorphin) [Cleaved   into: Alpha-neoendorphin; Beta-neoendor- phin; Big dynorphin (Big Dyn); Dynorphin  A(1-17) (Dyn-A17) (Dynorphin A); Dynorphin   A(1-13); Dynorphin A(1-8); Leu-enkephalin;   Rimorphin (Dynorphin B) (Dyn-B) (Dynorphin   B(1-13)); Leumorphin (Dynorphin B-29)] P24514 Chorion protein S18 SSVAGVAKKGYRK 0.35 4 1.42 9.82 429 W9R9I1 Uncharacterized protein RRLLEAAKEIVSLMHK 0.56 2.5 1.41 5.9 430 G4LYD1 MEG-5 KTLGTAFKTLLHNLWDLLKQ 0.56 2.5 1.40 8.18 431 R7ULK2 Uncharacterized protein (Fragment) LIKRFSTLWRDIWQVASNF 0.70 2 1.40 5.75 432 P55030 Interleukin-4 (IL-4) (B-cell stimulatory  LKDFLERLKAIMQK 0.70 2 1.40 9.57 433 factor 1) (BSF-1) (Lymphocyte stimulatory factor 1) Q3ECL0 Protein RALF-like 9 ANPYQRGCEKINRCR 0.46 3 1.39 9.3 434 B4LKD2 GJ20154 CRVARDFLRECMQHLKY 0.56 2.5 1.39 5.49 435 O95968 Secretoglobin family 1D member 1 KTLGKIAEKCDR 0.69 2 1.39 9.47 436 (Lipophilin-A) Q2V392 Putative defensin-like protein 25 QRCNRWCHNGCGNGKG 0.40 3.5 1.39 8.39 437 J7HBR9 Maxadilan related protein SKAKDAIAGLFTKAKSAL 0.46 3 1.38 9.78 438 KDVL M3Z119 Interleukin KEFLERLKSLIQRMI 0.69 2 1.38 9.37 439 Q9TV67 Interferon gamma (IFN-gamma) RKAISELIRVMKDL 0.69 2 1.38 9.8 440 Q22T52 Transmembrane protein, putative KTIQSWLNKFLSCLHI 0.55 2.5 1.38 7.7 441 P0C5M1 Uncharacterized protein YDR371C-A MSAIVKVVSNIIRPL 0.69 2 1.38 9.99 442 D3GGX2 Interleukin QEFLNSFSKLMQKLFKN 0.68 2 1.37 9.32 443 Q58IU6 Interleukin QEFLNSFSKLMQKLFKN 0.68 2 1.37 9.3 444 L7MA95 Putative secreted peptide ADKCLRYLLKNI 0.68 2 1.37 9.59 445 A0CK36 Chromosome undetermined scaffold_2, FKNCVKNILKDCQT 0.68 2 1.36 5.87 446 whole genome shotgun sequence A0A061J336 Pterin-4-alpha-carbinolamine dehydratase LARRMNEVFKEMLRP 0.68 2 1.36 6.19 447 C4R2P5 Long chronological lifespan protein 2 CKWVEKAWKGLL 0.68 2 1.35 5.24 448 P05105 Lysozyme (EC 3.2.1.17) (1,4-beta-N- ISVAATCAKKIYK 0.45 3 1.35 8.8 449 acetylmuramidase) F4PF44 Putative uncharacterized protein (Fragment) NELKELVKKASDIIKK 0.67 2 1.34 5 450 P14730 WAP four-disulfide core domain protein  NIQKCCSNGCGHVCK 0.54 2.5 1.34 8.77 451 18 (Extracellular peptidase inhibitor)  (Protein WDNM1) (Fragment) H3GQQ3 Uncharacterized protein NGLLQRIQSWWKNLFQHGAS 0.54 2.5 1.34 9.29 452 V9NJM6 IL-4 delta 3 TTLKDFLENLKRIMQK 0.67 2 1.34 8.85 453 A7KH73 Nodule Cysteine-Rich (NCR) secreted FREIPQCINSICKCMKG 0.67 2 1.34 6.5 454 peptide (Nodule-specific cysteine-rich  peptide 54) (Uncharacterized protein) P48617 Erythropoietin DALSKLFRIYSNFLRG 0.67 2 1.33 8.68 455 P33709 Erythropoietin DALSKLFRIYSNFLRG 0.67 2 1.33 8.68 456 Q6QN06 Matrix Gla protein (MGP) GYNAAYNRYFRK 0.44 3 1.33 9.39 457 P08493 Matrix Gla protein (MGP) (Cell growth- GYNAAYNRYFRK 0.44 3 1.33 8.66 458 inhibiting gene 36 protein) W7XBW1 Uncharacterized protein DSIFSKMYNCWRKCA 0.67 2 1.33 6.29 459 P33622 Apolipoprotein C-III (Apo-CIII) (ApoC-III) FRFLKGYWSKFTD 0.66 2 1.33 4.35 460 (Apolipoprotein C3) W5U2R0 Pro-sepiatocin QELFSLLKRLINKVN 0.66 2 1.32 8.35 461 W5U1W8 Sepiatocin QELFSLLKRLINKVN 0.66 2 1.32 7.92 462 Q4TJ01 Chromosome undetermined SCAF1215, whole KLIDTVIKQLRNLIAT 0.66 2 1.32 10.1 463 genome shotgun sequence G1QEP5 Uncharacterized protein (Fragment) LLEKIKTCWNKILSGT 0.66 2 1.32 8.48 464 P0C7P5 Bradykinin-potentiating and C-type  GGGGGGGGGARRMKGLAKKAM 0.26 5 1.32 11 465 natriuretic peptides (BPP-CNP) [Cleaved  into: Bradykinin-potentiating peptide Tf1; Bradykinin-potentiating peptide Tf2; Bradykinin-potentiating peptide Tf3; C- type natriuretic peptide Tf-CNP; C-type  natriuretic peptide Tf-CNP(3-22); C-type  natriuretic peptide Tf-CNP(6-22)] B7EY37 cDNA clone:001-127-H01, full insert AAWFQFFNRFLKYITSL 0.66 2 1.32 11.79 466 sequence G5ASQ0 Interleukin-4 TTLKDFLENLKTILKK 0.66 2 1.31 8.78 467 Q9FHA6 Protein RALF-like 34 VHPYSRGCSSITRCR 0.37 3.5 1.31 10.63 468 F7GL96 Uncharacterized protein (Fragment) LQSILELVHRVLRHLAQ 0.65 2 1.30 7.89 469 H9KY95 Uncharacterized protein (Fragment) LQSFLELVHRVLRHLAQ 0.65 2 1.30 6.4 470 Q03461 Non-specific lipid-transfer protein 2 CLKSAANAIKGI 0.65 2 1.30 9.16 471 (LTP 2) V4MH62 Uncharacterized protein SVYAQLSSVARTMIKRLEHFI 0.52 2.5 1.30 8.52 472 P63291 Vasoactive intestinal peptide (VIP) AVKKYLNSILN 0.65 2 1.29 9.82 473 (Vasoactive intestinal polypeptide) P63290 Vasoactive intestinal peptide (VIP) AVKKYLNSILN 0.65 2 1.29 9.82 474 (Vasoactive intestinal polypeptide) P84488 Vasoactive intestinal peptide (VIP) AVKKYLNSILN 0.65 2 1.29 9.82 475 (Vasoactive intestinal polypeptide) P63289 Vasoactive intestinal peptide (VIP) AVKKYLNSILN 0.65 2 1.29 9.82 476 (Vasoactiveintestinal polypeptide) V5HF12 Putative secreted protein QVGQILRECAKNLKN 0.65 2 1.29 4.75 477 P01581 Interferon gamma (IFN-gamma) HKAVNELIRVIHQL 0.65 2 1.29 9.48 478 L7M2P0 Uncharacterized protein RLLHGLLHSLLHSFSH 0.23 5.5 1.29 9.84 479 KFLDSFMRHMCTVCRNM D8TM42 Putative uncharacterized protein YWIHMLRYVAKT 0.52 2.5 1.29 9.58 480 P13589 Pituitary adenylate cyclase-activating  AVKKYLAAVLGK 0.43 3 1.29 5.61 481 polypeptide (PACAP) [Cleaved into: PACAP- related peptide (PRP-48); Pituitary   adenylate cyclase-activating poly-   peptide 27 (PACAP-27) (PACAP27); Pitui- tary adenylate cyclase-activating poly- peptide 38 (PACAP-38) (PACAP38)] P41535 Pituitary adenylate cyclase-activating AVKKYLAAVLGK 0.43 3 1.29 5.57 482 polypeptide (PACAP) [Cleaved into: PACAP- related peptide (PRP-48); Pituitary   adenylate cyclase-activating poly-   peptide 27 (PACAP-27) (PACAP27); Pitui-   tary adenylate cyclase-activating poly- peptide 38(PACAP-38) (PACAP38)] P16613 Pituitary adenylate cyclase-activating  AVKKYLAAVLGK 0.43 3 1.29 5.54 483 polypeptide (PACAP) [Cleaved into: PACAP-  related peptide (PRP-48); Pituitary   adenylate cyclase-activating poly- peptide 27 (PACAP-27) (PACAP27); Pitui-  tary adenylate cyclase-activating poly- peptide 38 (PACAP-38) (PACAP38)] P18509 Pituitary adenylate cyclase-activating  AVKKYLAAVLGK 0.43 3 1.29 5.38 484 polypeptide (PACAP) [Cleaved into:   PACAP-related peptide (PRP-48);   Pituitary adenylate cyclase-activating polypeptide 27 (PACAP-27) (PACAP27); Pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38) (PACAP38)] O70176 Pituitary adenylate cyclase-activating  AVKKYLAAVLGK 0.43 3 1.29 5 485 polypeptide (PACAP) [Cleaved into: PACAP-  related peptide (PRP-48); Pituitary aden- ylate cyclase-activating polypeptide 27   (PACAP-27)(PACAP27); Pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38) (PACAP38)] Q29W19 Pituitary adenylate cyclase-activating  AVKKYLAAVLGK 0.43 3 1.29 4.91 486 polypeptide(PACAP) [Cleaved into: PACAP- related peptide (PRP-48); Pituitary aden- ylate cyclase-activating polypeptide 27    (PACAP-27) (PACAP27);Pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38) (PACAP38)] P41534 Glucagon family neuropeptides [Cleaved  AVKKYLAAVLGK 0.43 3 1.29 9.4 487 into: Growth hormone-releasing factor 1-46  (GRF) (Growth hormone-releasing hormone)  (GHRH); Pituitary adenylate cyclase-acti- vating polypeptide 27 (PACAP-27)(PACAP27);   Pituitary adenylate cyclase-activating   polypeptide 38 (PACAP-38) (PACAP38)] P41585 Glucagon family neuropeptides [Cleaved  AVKKYLAAVLGK 0.43 3 1.29 9.31 488 into: Growth hormone-releasing factor   (GRF) (Growth hormone-releasing hormone)   (GHRH); Pituitary adenylate cyclase-  activating polypeptide (PACAP)] Q09169 Glucagon family neuropeptides [Cleaved  AVKKYLAAVLGK 0.43 3 1.29 6.28 489 into: Growth hormone-releasing factor (GRF)  (Growth hormone-releasing hormone) (GHRH); Pituitaryadenylate cyclase-activating polypeptide 27 (PACAP-27) (PACAP27); Pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38) (PACAP38)] V9LKT2 Interleukin LKKFLQFIYYCC 0.64 2 1.28 5.61 490 D0N540 Secreted RxLR effector peptide protein,  LSDAKQWIKTMFKNW 0.64 2 1.28 5.96 491 putative (Fragment) C9X4J0 Bradykinin-potentiating peptide (BPP) AKGKQMLKEYANKV 0.43 3 1.28 10 492 (TdBPP) P42692 Somatoliberin (Growth hormone-releasing GMFNKAYRKALGQLSA 0.43 3 1.28 9.31 493 factor) (GRF) (Growth hormone-releasing  hormone) (GHRH) A8MQI8 Protein RALF-like 5 GGLSTWKKLLDTILKIP 0.64 2 1.27 9.79 494 A2ZAS9 Non-specific lipid-transfer protein 3 CLKNMASSFRNLN 0.63 2 1.27 8.92 495 (LTP 3) Q2QYL3 Non-specific lipid-transfer protein 3 CLKNMASSFRNLN 0.63 2 1.27 8.92 496 (LTP 3) L0GB04 La1-like protein 15 VTCASQALKRGCKSV 0.42 3 1.27 9.03 497 G1TIN8 Uncharacterized protein CSSWPHLLREILRAARL 0.51 2.5 1.27 7.83 498 O35735 Interferon gamma (IFN-gamma) RKAVSELKKVMNDLL 0.63 2 1.27 9.84 499 B4LY36 GJ24431 GQVRNFGNACEKIVHSCKTG 0.51 2.5 1.26 5.29 500 Q99935 Opiorphin prepropeptide (Basic proline-   AFKSFWQKLFAIFG 0.63 2 1.26 10.7 501 rich lacrimal protein) (Proline-rich pro- tein 1) (PRL1) [Cleaved into: Opiorphin] P02659 Apovitellenin-1 (Apo-VLDL-II) (Apo-II) ARLTKLAEQLMEKIKNLC 0.63 2 1.26 9.21 502 (Apovitellenin I) (Very low density lipo- protein II) Q9YGI2 Probable weak neurotoxin NNAM1 GKRYIRGCADTC 0.63 2 1.26 8.99 503 O49320 Protein RALF-like 18 NKYRRGCSAATGCYRF 0.32 4 1.26 9.7 504 Q29146 Beta-lactoglobulin (Beta-LG) NKGMEEFKKIVRTLT 0.63 2 1.26 4.91 505 Q9TWH3 Venom peptide isomerase light chain VATVKNCGKKLLAT 0.42 3 1.26 9.79 506 A1YEU0 IL10 (Fragment) NMLRDLRDAFSRVKTFF 0.63 2 1.26 7.99 507 Q6LBF4 Interleukin-10 (Fragment) NMLRDLRDAFSRVKTFF 0.63 2 1.26 7.97 508 Q9BDX4 Interleukin-3 (IL-3) (Hematopoietic growth RKLKKYLEALDNFLNF 0.63 2 1.26 5.45 509 factor) (Mast cell growth factor) (MCGF)  (Multipotential colony-stimulating factor)  (P-cell-stimulating factor) Q29FI2 GA12182 KHAHSMITSMLTFVSSVM 0.42 3 1.26 9.52 510 NFGRSFVKD B4H186 GL22527 KHAHSMITSMLTFVSSVM 0.42 3 1.26 9.52 511 NFGRSFVKD O77559 ADM [Cleaved into: Adrenomedullin (AM); RAHQVLANLLKM 0.50 2.5 1.26 11 512 Proadrenomedullin N-20 terminal peptide  (ProAM N-terminal 20 peptide) (PAMP)  (ProAM-N20)] O97944 Alpha-S2-casein QKFLNKIYQYYQTFL 0.63 2 1.25 5.8 513 Q8WT56 Fatty-acid and retinol-binding protein 1 EKFKRIANSFLQ 0.63 2 1.25 8.81 514 (Ls-FAR-1) Q25619 Fatty-acid and retinol-binding protein 1  EKFKRIANSFLQ 0.63 2 1.25 8.62 515 (Antigen maltose-binding protein) (Ov-  FAR-1)(Ov20) (OvMBP/11) (OvS1) (S1 protein) O43555 Progonadoliberin-2 (Progonadoliberin II) RHLARTLLTAARE 0.42 2.5 1.06 10.8 516 [Cleaved into: Gonadoliberin-2   (Gonadoliberin II) (Gonadotropin-releas-   ing hormone II) (GnRH II) (Luliberin II)   (Luteinizing hormone-releasing hormone    II) (LH-RH II);GnRH-associated peptide 2  (GnRH-associated peptide II)] K9M1U5 Interferon lambda-4 (IFN-lambda-4) CARLRHVARGIADAQAVL 0.36 2.5 0.89 11.3 517 SGL

When assessed against a panel of prototypic and important human pathogens, it was found that the study test peptides had potent activity against nearly all of the organisms considered (FIG. 8). In particular, of the interleukins, sequences derived from turkey IL-5 (tIL-5) were markedly potent against all organisms at pH's representative of physiologic (pH 7.5) and septic/wound (pH 5.5) environments. Sequences derived from human interleukins (hIL-7, hIL-13, hIL-21) were somewhat less potent at pH 7.5, though retained significant activity at the physiologically important pH 5.5. Likewise, the peptides derived from human and bat IFN-γ ad significant activity against most organisms, being somewhat more potent at pH 7.5 than 5.5. Of the study test peptides, perhaps some of the most impressive were derived from Phytophthora. While Pp-1 and Pp-4 had significant activity against many organisms, Pp-2 was particularly potent against bacteria and fungi, activities that were retained at pH 5.5.

This example has utilized an alternate computational approach to screen for conserved elements common to a singular class of AHAPs as a means towards discovering universal sequence and structural elements that may confer microbicidal activity. Here, this example first identified a consensus sequence that was common to a majority of known AHAPs. This consensus was then subjected to an iterative refinement, based on alignments with both sequence and structure databases, to generate a linear formula that described an amphipathic helical span that was representative of most known AHAPs. In particular, this sequence formula consisted of a 18 residue span that included the following polar [EDKRHQNTS(AG)] and non-polar [VMCILFWY(AG)] residues. When translated into a three dimensional radial array, this α-core formula describes an idealized amphipathic helix comprised of equivalent polar and non-polar facets (FIGS. 1A-B and 2).

One component of the iterative optimization of the α-core formula involved queries against a control AHAP dataset. These studies revealed that glycine and alanine were critical elements found within AHAP amphipathic domains, as preliminary versions of the formula lacking these residues were largely unsuccessful, retrieving fewer than 10% of control AHAP sequences. Given the inherent amphipathic frequency of the α-core formula, it was possible to assess the positional requirement for specific residues on either the polar or non-polar face of an amphipathic helix. Initial studies with glycine indicated that it was essential that this residue be included with both the polar and non-polar residue search terms, as omission from either group dramatically reduced the number of returned sequences from the control dataset. Likewise, similar studies with alanine, revealed that it was important to include this residue with both the polar and non-polar residue groups to efficiently retrieve most AHAPs from the control dataset. As the α-core formula is translated into a three dimensional helical array, these studies further indicated that alanine and glycine are likely to be found on either the polar or non-polar face of AHAP amphipathic helices.

Similar to the above sequence-based optimization studies, structure-based alignments with the PDB database were also carried out. These studies revealed that while the inclusion of proline in the formula dramatically reduced the number of retrieved helices, glycine and alanine were tolerated and were commonly found in helical spans. These studies further revealed that the α-core sequence formula retrieved helical spans with a high degree of fidelity, correctly identifying such domains more than 90% of the time. While the α-core formula was not designed as a secondary structure characterization tool, its efficiency in identifying helical spans compares favorably with such tools which correctly identify helices with frequencies ranging from xx to 81.5%.

Database Searches

Once the α-core formula was refined, it was used as a query against the SwissProt database. These studies revealed that it retrieved nearly all classes of AHAPs (94%) and approximately 89% of all known individual AHAPs with a relatively high degree of specificity. Upon examination, those individual peptides or peptide families that were not retrieved by the formula were often found to have relatively short stretches of amphipathicity or interrupted helical domains, such as the “ranabox” peptides of amphibians which are characterized as having a C-terminal helix-turn-helix domain.

It is noteworthy that while the formula describes an idealized perfectly amphipathic helix, it successfully retrieved nearly all classes, and most individual AHAPs. This suggests that nearly all AHAPs have a span of near perfect amphipathicity for at least a 12 residue span representing the size of the domain queried in these studies. Given this observation, it supports the current hypothesis that amphipathicity is a critical component of microbicidal activity and is likely essential for the membrane permeabilizing properties of AHAPs.

Residue Frequency within AHAP Helices

As the α-core sequence formula returns aligned datasets, it was possible to assess the relative frequency of various residues within AHAP α-helical spans. Moreover, these data allowed for some generalizations to be made regarding universal features common to AHAP helices. Overall, beyond the inherent amphipathicity found within AHAPs, these data revealed that several residues were strongly favored within amphipathic helices derived from various classes of organisms.

Glycine

One notable finding from these studies was that glycine is highly represented in AHAP helices from arthropods and amphibians (30-35% of all residues), whereas it occurs less frequently in mammals (15% of all residues).

Glycine in AHAP Helices of Arthropods and Amphibians

The presence of glycine within a majority of known AHAP helices would not initially be expected as this residue is known to destabilize α-helical structures. However, as was demonstrated by a large body of empirical evidence, many α-helical peptides are unstructured in aqueous environments, only to adopt an α-helical conformation in hydrophobic milieus. While the mechanism by which this occurs is not known, it is possible that peptides may become organized at the target as the relatively electronegative microbial surface may help to orient the cationic and other polar residues to one face of the amphipathic helical structure. In a similar fashion, the hydrophobic interior of the microbial plasma membrane may also help to organize the random coil structure by selectively interacting with hydrophobic residues within the peptide.

This lack of amphipathic organization prior to interaction with the target may be an important mechanism behind the selective toxicity of AHAPs that allows them to limit activity towards the host while still being able to adopt a microbicidal conformation as necessary when interacting with the target microbe.

Glycine in AHAP Helices of Higher Eukaryotes

One caveat regarding the presence of glycine in AHAP helices is that while it is common in the short peptides found within lower organisms, it occurs less frequently in the longer AHAPs of higher eukaryotes as well as in proteins with mixed domain architectures such as the chemokines, granulysin and NK lysin. If the supposition that glycine is an important means to keep AHAPs in an unstructured and inactive state prior to interaction with their microbial target, the lack of glycine in these larger peptides suggests that they may use an alternate means of microbicidal dampening to limit toxicity towards the host.

Cationic Residues in AHAP Helices of Arthropods and Amphibians

Beyond demonstrating an abundance of glycine in AHAP helices, residue frequency studies also revealed that lysine is highly favored over arginine in the amphipathic helices of the AHAPs considered in this study. Lysine was the most abundant cationic residue in study AHAP helices, where it was strongly preferred over arginine in non-mammalian peptides at a 12:1 ratio. In mammals, lysine was still preferred over arginine, but to a lesser extent with a 3:1 ratio. By comparison, lysine was less abundant in toxins where it was preferred over arginine at a 2:1 ratio.

It may be of some relevance that biophysical measures have demonstrated lysine is less efficient at generating membrane destabilizing NGC than arginine, as it can only interact with a single lipid head group at a time. Because of this, AHAPs that are rich in lysine often compensate for this lack of permeabilizing ability by having an increased mean hydrophobicity, in what has been termed the ‘saddle-splay curvature selection’ rule. In the current study, AHAP helices largely support this rule with the finding that, as the relative abundance of lysine is increased (N_(K)/N_(K)+N_(R)), mean peptide hydrophobicity is also increased.

Biophysical Properties of AHAP Helices as Compared with Other α-Helical Domains

Beyond measurements of residue frequency, a number of additional properties of helices retrieved by the α-core formula were determined including: Q, μH, H. When averaged across families, these quantitative data revealed a number of insights regarding the biophysical properties of retrieved AHAP helices versus those of toxins and other groups.

One defining characteristic of AHAPs is that they are typically cationic in nature, a property that has been shown to enhance their selectivity towards the relatively electronegative surface of many microorganisms. The universality of this biophysical property is supported by findings from the current study, where amphipathic helices derived from AHAPs had an average net charge of +2.0 over a twelve residue span. Moreover, if only cationic AHAPs are considered (Q≥0.5 for 707 of 803 retrieved sequences), this value rises to +2.7. By comparison the average charge for toxin and other helices retrieved in these studies was +1.3 and +0.6 respectively, suggesting that cationicity may be selectively favored in AHAPs as compared with other amphipathic peptides.

Another characteristic feature of AHAPs is that they are frequently amphipathic in nature, a property that has been deemed essential for their membrane permeabilizing activities towards microbes. As the α-core formula searches for amphipathic sequences, all of the helices returned by these queries were a priori amphipathic in nature. However, it was still of interest to compare the relative amphipathicity, as quantitated by μH, of identified helices between various classes of peptides. Results from the current study support the theory that amphipathicity is an essential biophysical property of AHAPs as the average hydrophobic moment for study AHAP helices was 0.52, a relatively high value when compared to archetypal amphipathic helices such as cecropin A (μH_(max)=0.61) or LL-37 (μH_(max)=0.78). In comparison, the amphipathicity of helices derived from toxins and other proteins were somewhat lower at μH=0.42 and 0.39 respectively.

In addition, mean hydrophobicity (H) was somewhat greater for AHAPs (0.42) than for toxins (0.34) or other study peptides (0.39). This observation suggests that a moderate level of hydrophobicity may be essential for the membrane permeabilizing properties of AHAPs.

Identify New Antimicrobial Sequences

The α-core sequence formula also retrieved a large number of uncharacterized sequences as well as proteins with an alternate primary function. Given the fidelity of retrieving antimicrobial sequences, it was of interest to determine whether some of these alternate sequences might also possess antimicrobial properties. As described, putative microbicidal sequences were scored for properties that were most discriminative at separating AHAPs from other sequences (μH*Q or μH*PI) and lead candidates of biological interest were synthesized for further study.

Representative peptide helices from three separate high-scoring groups, interleukins (tIL-5, hIL-7, hIL-13 and hIL-21), interferons (bIFN-γ, hIFN-γ) and uncharacterized sequences from P. parasitica were synthesized and tested for their microbicidal activity. Notably, all of these peptides displayed potent microbicidal activity, which in many cases was greater than that of classic AHAPs such as LL-37. These data strongly suggest that the amphipathic sequence formula can successfully identify amphipathic helices with antimicrobial properties.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The disclosures illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.

Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains. 

1. An isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14, and an amino acid derived from anyone of SEQ ID NO: 1-14 with one amino acid substitution, wherein the peptide is not longer than 100 amino acid residues in length.
 2. The isolated peptide of claim 1, wherein the peptide has antimicrobial activity.
 3. The isolated peptide of claim 1, wherein the peptide comprises the amino acid sequence of anyone of SEQ ID NO: 1-14.
 4. The isolated peptide of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 13 or
 14. 5. The isolated peptide of claim 1, wherein the peptide is not longer than 75 amino acid residues in length.
 6. The isolated peptide of claim 1, wherein the peptide is not longer than 60 amino acid residues in length.
 7. An isolated peptide comprising an amino acid sequence of SEQ ID NO:19-6860 or an amino acid derived from a sequence of SEQ ID NO:19-6860 with one amino acid substitution, wherein the peptide is not longer than 100 amino acid residues in length.
 8. A fusion peptide comprising a first fragment comprising the peptide of claim 7, and a second fragment having antimicrobial activity, wherein the fusion peptide is not longer than 100 amino acid residues in length.
 9. The fusion peptide of claim 8, wherein the second fragment comprises a gamma-core motif comprising two anti-parallel β-sheets interposed by a short turn region with a GXC or CXG sequence pattern integrated into one of the β-sheets.
 10. The fusion peptide of claim 9, wherein the gamma-core motif comprises CPTAQLIATLKNGRKICLDLQ (SEQ ID NO: 15) or a first amino acid sequence having at least 85% sequence identity to SEQ ID NO:
 15. 11. The peptide of claim 1, comprising one or more non-natural amino acid residues.
 12. A composition comprising the peptide of claim 1, and a pharmaceutically acceptable carrier.
 13. The composition of claim 12, further comprising an antimicrobial agent.
 14. The composition of claim 13, wherein the antimicrobial agent is selected from the group consisting of imipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycin and daptomycin.
 15. A method of treating an infection in a patient in need thereof, comprising administering to the patient an effective amount of the composition of claim
 12. 16. The method of claim 15, wherein the infection is caused by a Gram-negative bacterium, a Gram-positive bacterium or a fungus.
 17. A computer-implemented method of identifying a peptide having antimicrobial activity, comprising: identifying a consensus formula from aligned amino acid sequences known to have an antimicrobial activity; tuning the consensus formula with a test search against a plurality of proteins with known antimicrobial activity; and searching in a protein database, with one or more processors, for amino acid fragments matching the consensus formula, wherein the search takes as input one or more criteria selected from the group consisting of location of the fragment in a protein, size of the protein, organism of the protein, and signal peptide of the protein.
 18. The method of claim 17, wherein the tuning comprising shortening the length of the consensus formula or changing substation options at one or more amino acid residues.
 19. (canceled) 